MICROORGANISMS AND METHODS IN THE FERMENTATION OF

BENZYLISOQUINOLINE ALKALOIDS

SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted

electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on August 16, 2018, is named INX00389_SL.txt and is 210,766 bytes in size.

BACKGROUND OF THE DISCLOSURE

[0002] Benzylisoquinoline alkaloids ("BIAs") are a large and structurally diverse family of plant secondary tyrosine metabolites that exhibit a wide range of pharmacological activities.

Thebaine, a chemical compound also known as paramorphine and codeine methyl enol ether, belongs to the BIA class of compounds, and within that class, to a BIA subclass of compounds known as morphinan alkaloids, and has long been recognized as a useful feedstock compound in the manufacture of therapeutic agents, including, for example, morphine and codeine. Other BIAs that can be manufactured can include but are not limited to oripavine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxy codeinone and neopinone. Currently thebaine and other BIAs may be harvested from natural sources, such as opium poppy capsules (see e.g. , U.S. Pat. Appl. Pub. No 2002/0106761; see also e.g. , Poppy, the genus Papaver, 1998, pp 113, Harwood Academic Publishers, Editor: Bernath, J.). Alternatively, thebaine may be prepared synthetically. The latter may be achieved by a reaction sequence starting with ketalization of iodoisovanillin (see e.g. , Rinner, U. and Hudlicky, T., 2012, Top. Cur. Chem. 309; 33-66; Stork, G., 2009, J. Am. Chem. Soc. 131 (32) pp 11402-11406).

[0003] Salutaridine is an alkaloid that is a part of the morphinan alkaloid pathway. Salutaridine is formed by the enzymatic conversion of (R)-reticuline by salutaridine synthase (Sal Syn). Salutaridine is further converted to salutaridinol by the enzyme salutaridine reductase (SalR). Salutaridinol is converted to salutaridinol-7-O-acetate through the enzyme salutaridinol 7-0- acetyltransferase (SalAT). Salutaridmol-7-O-acetate is converted to thebaine through a spontaneous reaction or through the use of a thebaine synthase (THS).

[0004] The existing manufacturing methods for BIAs, including thebaine and other morphinan alkaloids and their derivatives, suffer from low yields and/or are expensive. Some of the known methodologies for the manufacture of thebaine exist in the production of undesirable quantities of morphinan alkaloid by-products (see e.g. , Rinner, U., and Hudlicky, J., 2012, Top. Cur. Chem. 209: 33-66). No methods exist to commercially biosynthetically manufacture BIAs, including thebaine and other morphinan alkaloids and their derivatives. Therefore, there is a need for efficient methods to synthesize BIAs including but not limited to reticuline, thebaine, morphine, oripavine, oxycodone, hydrocodone, oxymorphone, hydromorphone and others.

INCORPORATION BY REFERENCE

[0005] All publications, patents, and patent applications herein are incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference by its entirety. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.

SUMMARY

[0006] This application discloses microorganisms that are capable of producing alkaloids (e.g., benzylisoqumoline alkaloids ("BIAs") or any BIA intermediate, in an efficient manner, as well as methods of increasing the efficiency of BIA synthesis. The products that can be made by the processes and microorganism described herein can include, but are not limited to reticuline, salutaridine, salutaridinol, salutaridinol-7-O-aceteate, thebaine, morphine, oripavine, oxycodone, hydrocodone, oxymorphone, hydromorphone, or their derivatives.

[0007] Disclosed herein is a method of making salutaridine comprising contacting salutaridine synthase with reticuline within a medium to convert reticuline to salutaridine, where the pH of the medium is between 6 and 13. Also disclosed herein is a method of making

salutaridinol comprising contacting salutaridine reductase with salutaridine within a medium to convert salutaridine to salutaridinol, wherein the pH of the medium is between 5 and 13.

Further disclosed herein is a method of making salutaridinol-7-O-acetate comprising contacting salutaridinol 7-O-acetyltransferase with salutaridinol within a medium to convert salutaridinol to salutaridinol-7-O-acetate, wherein the pH of the medium is between 6 and 13. Also disclosed herein is a method of making thebaine comprising contacting thebaine synthase with to salutaridinol-7-O-acetate within a medium to convert to salutaridinol-7-O-acetate to thebaine, wherein the pH of the medium is between 5 and 13.

[0008] The methods described herein can also include the use of medium, where the medium does not contain any living cells. For example, the conversion of reticuline to salutaridine; salutaridine to salutaridinol; salutaridinol to salutaridinol-7-O-acetate; and/or salutaridinol-7-O- acetate to thebaine occurs outside of a cell, e.g., a cell-free system.

[0009] The methods described herein can also include the use of cell culture media. In some of these cases, the conversions, for example, of reticuline to salutaridine; salutaridine to salutaridinol; salutaridinol to salutaridinol-7-O-acetate; and/or salutandinol-7-O-acetate to thebaine occurs within a cell.

[00010] In some cases, when reticuline is used, the reticuline is R-reticuline. In some cases, the reticuline is S-reticuline. In some cases, the reticuline is both R-reticuline and S-reticuline.

[00011] The methods can include an adjustment of pH. For example, when using a salutaridine synthase, the pH of the media can be greater than 6.0. In some cases, the pH of the media is between 7 to 7.4. In some cases, the pH of the media is between 7.5 to 7.9. In some cases, the pH of the media is between 8 to 8.4.

[00012] In some cases, for example, when using a salutaridine reductase, the pH of the media can be greater than 5.0. In some cases, the pH of the media is between 5.5 to 5.9. In some cases, the pH of the media is between 6 to 6.4. In some cases, the pH of the media is between 6.5 to 7.0.

[00013] In some cases, for example, when using a salutaridinol 7-O-acetyltransferase, the pH of the media can be greater than 6.0. In some cases, the pH of the media is between 6.5 to 6.9. In some cases, the pH of the media is between 7 to 7.4. In some cases, the pH of the media is between 7.5 to 7.9.

[00014] In some cases, for example, when using a thebaine synthase, the pH of the media can be greater than 5.0. In some cases, the pH of the media is between 7 to 7.4. In some cases, the pH of the media is between 7.5 to 7.9. In some cases, the pH of the media is between 8 to 8.4.

[00015] The pH can be adjusted or maintained by supplementing said medium with an acidic or alkali substance or buffering reagent. For example, in some cases, the pH is maintained by supplementing the medium with an alkali substance. The alkali substance can be any alkali substance, such as NH ₄ OH or NaOH.

[00016] The contacting of the enzymes with their respective substrates can occur over a certain period of time. In some cases, the contacting of the enzymes with their respective substrates can be for at least 24 hours. In some cases, the contacting of the enzymes with their respective substrates can be for at least 48 hours. In some cases, the contacting of the enzymes with their respective substrates can be for between about: 24 and 48 hours. In some cases, the contacting of enzymes with their respective substrates can be for at least 30 seconds. In some cases, the contacting of enzymes with their respective substrates can be for at least 60 seconds.

[00017] When salutaridine is made, its presence of with the media can vary. In some cases, salutaridine can be present within the medium at a concentration of at least 75 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 200 μg/L. [00018] When salutaridmol is made, its presence of with the media can vary. In some cases, salutaridine can be present within the medium at a concentration of at least 25 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 100 μg/L.

[00019] When thebaine is made, its presence of with the media can vary. In some cases, thebaine can be present within the medium at a concentration of at least 750 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 900 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 1500 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 1900 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 15 μg/L. In some cases, thebaine can be present within the medium at a concentration of at least 25 μg/L.

[00020] When a cell is used to perform the reaction, the cell can be a yeast cell. The yeast cell can be from the genus Saccharomyces . For example, the yeast cell can be from the species Saccharomyces cerevisiae. In some cases, the cell can be a plant cell. In other cases, the cell can be a fungal cell. In some cases, the cell can be a bacterial cell.

[00021] The method, whether within a cell or outside of a cell, can comprise a salutaridine

synthase, salutaridine reductase, salutaridinol 7-O-acetyltransferase, and/or a thebaine synthase. The method, whether within a cell or outside of a cell, can also further comprises a purine permease and/or a cytochrome p450 reductase. If used within a cell, any one of these enzymes can be heterologous to the cell.

[00022] Also disclosed herein are vectors. In some cases, the vectors can comprise a nucleotide sequence that is substantially identical to any one of SEQ ID NOs. 58 to 63. In some cases, the vector can comprise a nucleotide sequence that is substantially identical to SEQ ID NO. 64. In some cases, the vector comprises a nucleotide sequence that is substantially identical to SEQ ID NO 65.

[00023] Further disclosed herein is a method of making a hydroxylated product comprising

contacting (7S)-salutaridinol 7-O-acetate with water where (7S)-salutaridinol 7-O-acetate is hydroxylated. The hydroxylated product can be any hydroxylated product described throughout the application, for example, including those in FIG. 2A.

[00024] In some cases, the method is performed within a cell. In such instances, the cell

sometimes (i) does not comprise thebaine synthase; (ii) comprises an inactive thebaine synthase; or (iii) comprises a thebaine synthase having reduced activity compared to a wild-type thebaine synthase. In some instances, the cell can also comprise a heterologous salutaridinol 7-0- acetyltransferase. Further, in some instances, the (7S)-salutaridinol 7-0-acetate used in the method is produced by a heterologous salutaridinol 7-O-acetyltransferase. The (7S)- salutaridinol 7-O-acetate used in the method, can in some cases, not come into contact with thebaine synthase. In some cases, the cell can further comprise an gene that is a tyrosine hydroxylase (TYR); DOPA decarboxylase (DODC); norcoclaurine synthase (NCS); 6-0- Methyltransferase (60MT); coclaurine N-methyltransferase (CNMT), cytochrome P450 N- methylcoclaurine hydroxylase (NMCH), and 4-0-methyltransferase (40MT); cytochrome P450 reductase (CPR), salutaridine synthase (SAS); salutaridine reductase (SalR); or any combination thereof. This additional gene(s) can be heterologous to the cell.

[00025] Further disclosed is a method of making thebaine comprising placing salutaridinol or salutaridinol 7-O-acetate in a pH of greater than 7.5 and maintaining the pH of greater than 7.5 until an S _N 2' mechanism takes place. In some cases, the method uses salutaridinol 7-O-acetate. Additionally, the method sometimes can take place within a cell. The pH used in the method can be greater than 8.0. In some cases, the method does not allow salutaridinol or salutaridinol 7-O-acetate to come in contact with water. For example, this can happen the method occurs within an enzyme. In some instances, the enzyme is thebaine synthase.

[00026] Also disclosed herein is a method of making a BIA comprising contacting (7S)- salutaridinol 7-O-acetate with an enzyme that is capable of converting (7S)-salutaridinol 7-O- acetate into a BIA, wherein said enzyme has a Vmax of greater than 2.0 nmol min ^"1 μg ^'1 . In some cases, the Vmax is between 1.5 to 4.0 nmol min ^"1 μg ^"1 . In other cases, the Vmax is greater than 4.0 nmol min ^"1 μg ^"1 . In some cases, the BIA is thebaine. In some cases, the pH used during the method is greater than 7.5.

BRIEF DESCRIPTION OF THE DRAWINGS

[00019] The novel features of the invention are set forth with particularity in the appended

claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[00020] FIGs. 1A, IB, and 1C. FIG. 1A shows the molecular pathway from glucose to L- tyrosine in yeast. FIG. IB shows the molecular pathway from glucose to L-tyrosine in bacteria such as E. coli. FIG. 1C shows the molecular pathway from L-tyrosine to BIAs (including but not limited to thebaine, morphine, and their derivatives).

[00021] FIGs. 2A to 2E. FIG. 2A depicts the thebaine pathway. Salutaridine is converted to (7S)-salutaridinol by salutaridine reductase (SalR). (7S)-Salutaridinol yields thebaine spontaneously at pH <5, but in the plant is 7-O-acetylated by salutaridinol 7-O-acetyltransferase (SalAT). (75)-Salutaridinol 7-O-acetate can spontaneously undergo allylic elimination yielding thebaine, but this reaction competes with its degradation to unstable hydroxylated byproduct(s) with ionic m/z 330. This byproduct is not detected in the plants due to the activity of thebaine synthase (THS). Further, hydroxylated by-products can be formed spontaneous from (7S)- Salutaridinol 7-O-acetate. If desired, these hydroxylated by-products can be used later to form useful products. FIG. 2B shows the results of a high-resolution mass spectral fragmentation analysis of alkaloid byproduct (m/z 330). The expanded region (m/z 300-340) highlights the occurrence of a fragment with m/z 312, possibly a dehydration product of the parent ion. FIG. 2C shows that the MS" analysis revealed similarities and differences between (75)-salutaridinol and the m/z 330-byproduct. MS" on the m/z 330-byproduct yielded ions shown on the right side. Although some byproduct ions were common with those obtained from similar (7S)- salutaridinol analysis (a, f, g), others (c, d, e, h) were not. Conversely, ions shown on the left side were unique to (7S)-salutaridinol MS" (i, j, k, I, m, n) and were not observed in the m/z 330- byproduct spectra. FIG. 2D shows a high-resolution fragmentation analysis of m/z 330- byproduct generated spontaneously by degradation (75)-salutaridinol 7-O-acetate. This spectrum represents an average of 161 individual scans captured over 5 minutes of continuous sample infusion (5 μΙνΊηίη). Ionization was performed by ESI at room temperature. Mass error was < 2 ppm across all MS" datasets, allowing reliable prediction of elemental formula. FIG. 2E depicts a mechanism upon which SalAT-catalyzes the formation of salutaridinol 7-O-acetate, and where an S _N 2' allylic elimination in the presence of water lead to the formation of a hydroxylated byproduct with m/z 330.

[00022] FIGs. 3A to 3C: FIG. 3A shows the time and pH dependence of thebaine formation from (7<S)-salutaridinol 7-O-acetate mediated by THS. Direct assays were performed using 40 μΜ and either 0.6 μg or 0.4 μg of purified recombinant THS2 at pH 7.0 (TOP) and pH 8.0 (BOTTOM), respectively. Values represent mean ± standard deviation of three replicates. FIG. 3B shows the dependence of THS2 activity on pH. Direct assays were conducted for 30 sec using 40 μΜ salutaridinol 7-O-acetate substrate, 0.6 μg of purified recombinant THS2, and pH- appropriate buffer as shown. FIG. 3C shows the sigmoidal dependence of THS2 activity on substrate concentration. Direct assays were conducted for 30 sec using variable concentrations of (75)-salutaridmol 7-<9-acetate, and either 0.6 μg (pH 7.0) (TOP) or 0.4 μg (pH 8.0)

(BOTTOM) of purified recombinant THS2. Values represent mean ± standard deviation of three replicates.

[00023] FIGs. 4A and 4B. FIG. 4A shows genotypes used in experiments in FIG. 4B. The strains contained up to nine chromosomally integrated plant genes encoding BIA biosynthetic enzymes capable of converting (S)-norlaudanosoline to salutaridine, salutaridinol 7-<3-acetate, or thebaine. Strain Sc-2 harbored the first seven biosynthetic genes, resulting in the production of salutaridine. Strain Sc-3 contained two additional genes, encoding SalR and SalAT, leading to the formation of salutaridinol 7-O-acetate, whereas strain Sc-4 also included SalR, SalAT and THS2 genes. FIG. 4B shows levels of reticuline, salutaridine, salutaridinol, and thebaine when the strains of FIG. 4A where cultured using standard protocols with the addition of 2.5 mM (R/S)-norlaudanosoline to the induction medium, followed by a 24, 48, and 96-hour fermentation at 30 C°. Values represent the mean ± standard deviation of at least 3 independently transformed yeast lines. pEV-1 represents a plasmid having an empty vector that was added to Strains Sc-2, Sc-3, and Sc-2. pTHS2 represents a plasmid that expresses a THS2 gene that was added to Strains Sc-2, Sc-3, and Sc-2.

[00024] FIG. 5 shows the kinetic constants for THS2. All data was acquired using the direct in vitro THS assay performed at pH 7.0 and 8.0. Values represent mean ± standard deviation of three replicates.

[00025] FIG. 6 shows the MRM transition of alkaloid standards. These standards are used in for MS ⁿ analysis. Quantifier MRM ion peak areas were compared to calibration curves of pure standards prepared in the appropriate matrix using MassLynx v4.1 for salutaridinol and thebaine and QuanLynx v4.1 for reticuline.

[00026] FIGs. 7A and 7B. FIG. 7A shows the BIA pathway from R-reticuline to thebaine with the intermediates salutaridine, salutaridinol, salutaridinol-7-O-actetate through the use of the enzymes salutaridine synthase (SalSyn), cytochrome P450 reductase (CPR), salutaridine reductase (SalR), salutaridinol 7-O-acetyltransferase (SalAT). FIG. 7B shows the experimental designed use to determine the effect of pH on production utilizing BIA pathway enzymes, such as Sal Syn, Sal R, and SalAT. Salutaridine, salutaridinol, and thebaine titers were measured in these experiments.

[00027] FIG. 8 shows salutaridine, salutaridinol, and thebaine titers based on fermentation in various pH ranges using MES buffer. SalSyn, SalR, SalAT, and CPR were all expressed in yeast and cultured in media supplemented with ImM R-reticuline. As shown, higher pH significantly improved the formation of salutaridine, salutaridinol, and thebaine compared to the water control and lower pHs.

[00028] FIGs. 9A and 9B shows salutaridinol and thebaine titers affected by pH when yeast expressing enzymes SalSyn, CPR, SalR, SalAT, and THS were grown in media supplemented with ImM R-Reticuline or ImM Salutaridine. Buffered pH at pH6.5 and pH 7.5 lead to an increase in both salutaridinol and thebaine titers at the measured time points (24 and 48 hours) compared to the unbuffered control.

[00029] FIG. 10 shows the difference in thebaine titers after culturing for 48 hours in either SE or YP medium. When starting from a reticuline feed, the strain grown in pH 7.5 and 6.5 exhibited increased thebaine titers (normalized to OD) when compared to negative unbuffered controls. However, strains grown in YPD media exhibited higher thebaine titers when compared to strains grow in SE media. These increased titers between different media were seen in all groups, including the negative control, pH 6.5 and pH 7.5. When starting from a salutaridine feed, the strain grown in pH 7.5 and 6.5 exhibited a significant increase in thebaine titers (normalized to OD) when compared to negative controls (having a pH of < 6.0). However, strains grown in SE media exhibited much higher thebaine titers (about 100% or more) when compared to strains grow in YPD media. These increased titers between different media were seen in all groups, including the negative control, pH 6.5 and pH 7.5.

[00030] FIG. 11 shows reticuline titers of four (4) strains cultured in three separate culture

conditions. The strains were transformed with enzymes as follows: Strain 1 : DODC to SalSyn; Strain 2: Strain 1 + SalR and SalAT; Strain 3: Strain 2 + BetVl-A; and Strain 4: Strain 2 + BetVl-B. The culture conditions were set as follows: 1) 24 hours (not buffered, having a pH of <6.0), 2) 48 hours (not buffered as a negative control), and 3) 48 hours (adjusted to a pH of 7.5 after 24 hours). All strains harvested at 24 hours (having a pH of < 6.0) produced similar reticuline titers between the various strains (Strains 1, 2, 3, and 4). The strains harvested at 48 hours (culture condition 2) produced similar reticuline titers. However, all strains that were cultured in pH 7.5 (culture condition 3) produced higher reticuline titers (of approximately 25% to 30%). Under culture condition 3, the BetVl-B strain produced slightly less reticuline than the other strains.

[00031] FIG. 12 shows titers of salutaridine, salutaridinol, and thebaine measured in these strains.

Strain 1 (strains having enzymes from DODC to SalSyn) produced very high levels of salutaridine both after 24 hours and 48 hours supplemented with water (negative control). However, after 48 hours at pH 7.5, strain 1 produced high levels of salutaridine, approximately 25% more than its negative control or after 24 hours. As expected, Strain 1 failed to produce any detectable levels of salutaridinol or thebaine, since the enzymes that perform those reactions were not present in the strain. Strain 2 (strains having enzymes from DODC to SalSyn, plus SalR and SalAT) showed elevated levels of salutaridine when cultured in pH 7.5 for 48 hours. Strains cultured for 24 hours (having a pH of < 6.0) and cultured for 48 hours diluted in water (negative control) demonstrated lower salutaridine levels (approximately 30% decrease). Strain 2 also produced salutaridinol and thebaine at similar levels at 24 hours, 48 hours (negative control) and 48 hours in pH 7.5. Strain 3 (strains having enzymes from DODC to SalSyn, plus SalR and SalAT plus BETV1-A) produced slightly elevated levels of salutaridine only when cultured at pH 7.5 after 48 hours. Salutaridinol levels were also mostly unchanged at 24 hours, 48 hours (negative control) and 48 hours in pH 7.5. However, thebaine levels increased

(approximately 40%) when culturing in pH 7.5 for 48 hours, compared to 24 hour culture and negative control (unbuffered) cultures. A fourth strain was created ("Strain 4"; a strain having enzymes from DODC to SalSyn, plus SalR and SalAT plus BETVl-B) produced slightly elevated levels of salutaridine only when cultured at pH 7.5 after 48 hours, (data not shown) Salutaridinol levels were also mostly unchanged at 24 hours, 48 hours (negative control) and 48 hours in pH 7.5. (data not shown) However, thebaine levels increased significantly

(approximately 30%) when culturing in pH 7.5 for 48 hours, compared to 24 hour culture and negative control cultures, (data not shown) Strain 4 produced the most overall thebaine levels after 48 hours when cultured at a pH of 7.5. (data not shown)

[00032] FIG. 13 shows that OD levels were similar between the same strains at 24 hours, 48 hours (no buffer), and 48 hours (with buffering - pH 7.5).

[00033] FIG. 14 shows that Strain: Y16_T7, which has the following integrated genes

Pbra60MT, CjapCNMT, Psom40MT, REPI-2, PbraSalSyn, PbraCPRl, PsomSalR, PsomSalAT, and PsomBetvl-1 produced higher titers of thebaine at pH 7.5 after 48 and 96 hours. The SE Media (also known as SD-MSG) contained 2.5mM Racemic NLDS for 96 hours. lOOmM HEPES (pH 7.5) was added at either 24 hours or 48 hours.

[00034] FIGs. 15A, 15B and 15C. FIG. 15A shows salutaridine titers using the yeast strain Y16_T9 (which has the following integrated genes Pbra60MT, CjapCNMT, Psom40MT, REPI-2, PbraSalSyn, PbraCPRl, PsomSalR, PsomSalAT that were further transformed with either Empty Vector (EV) or High copy plasmid containing HA-Bet l (N-terminal HA epitope tag). Levels of salutaridine increased by 4-fold at pH 7.5 compared to the unbuffered control or at pH 6.5. FIG. 15B shows thebaine titers at pH 6.5, pH 7.5, and unbuffered control. Thebaine production in the presence of Betvl, increased approximately 5-fold at pH 7.5 compared to pH 6.5 and unbuffered control. FIG. 15C shows m/z 330 levels at pH 6.5, pH 7.5, and unbuffered control, m/z 330 levels dropped approximately 50% in the presence of Betvl at pH 6.5, pH 7.5, and in unbuffered conditions.

[00035] FIGs. 16A and 16B. FIGs. 16A and 16B show two strains with different genotypes in differing pH conditions. Each strain contained three methyltransferases (60MT, CNMT, and 40MT), one NMCH, two variants of SalSyn, and a reticuline epimerase (REPI). In addition, the APY254 strain expressed a P. somniferum CPR and a second copy of NMCH, whereas the APY299 expressed an A. annua CPR. FIG. 16A shows that when fed with either NCC or NLDS without buffer, both strains produced similar levels of combined reticuline and salutaridine and less than half was salutaridine. However, when the media was buffered to pH 7.5 with HEPES, total production from an NCC feed increased up to 14x and the majority of the product was salutaridine (FIG. 16B). Production from an NLDS feed also increased up to 3x with a similar increase ratio of salutaridine to reticuline (FIG. 16B).

[00036] FIGs. 17A to 17C. FIG. 17A shows the pH of the media present when fermenting strain yGPVR151 in the presence or absence of YP over a 136 hour period. pH was kept over pH 4.0 at all times. NH ₄ OH was incrementally added to the media when the pH reached 4.0 to raise the pH to a setpoint of 6.0. FIG. 17B shows the levels of dopamine at various time points for the same culture over a 136 hour period. FIG. 17C shows the levels of reticuline at various time points for the same culture over a 136 hour period.

[00037] FIGs. 18A to 18D. FIG. 18A shows the pH of the media present when fermenting strain yGPVR251 over a 120-140 hour period. Four test groups were used: 1) 7% ρ(¾ and no pH regulation; 2) 20% p0 ₂ and no pH regulation; 3) 20% p0 ₂ and pH regulated at 6.0; and 4) 20% pC" ₂ and no pH regulation. FIG. 18B shows the levels of dopamine for the same culture over a 136 hour period. FIG. 18C shows the levels of reticuline for the same culture over a 136 hour period. FIG. 18D shows the levels of salutaridine for the same culture over a 136 hour period.

[00038] FIGs. 19A to 19D. FIG. 19A shows the pH of the media present when fermenting strain yGPVR353 or yGPVR352 over a 115 hour period. Four test groups were used: l) yGPVR353 with pH regulated at 6.5; 2) yGPVR353 with pH regulated at 6.0; 3) yGPVR353 with pH regulated at 6.5; and 4) yGPVR352 with pH regulated at 6.0. The pH of the media for all four test groups were increased to over pH 8.0 at around 24 hours in the fermentation. FIG. 19B shows the levels of dopamine for the same culture over a 136 hour period. FIG. 19C shows the levels of reticuline for the same culture over a 136 hour period. FIG. 19D shows the levels of salutaridine for the same culture over a 136 hour period.

[00039] FIGs. 20A to 20D. FIG. 20A shows the pH of the media present when fermenting strain yGPVR454 over a 115 hour period. pH was regulated with acids and bases from both sides to keep pH at a constant 6.0. Two test groups were used with repeats: 1) yGPVR454 with no pH regulation (yGPVR454 normal pr); 2) yGPVR454 with no pH regulation (yGPVR454 normal pr); 3) yGPVR454 with pH regulation at 6.0 (yGPVR454 L-DOPA in HCl); and 4) yGPVR454 with pH regulation at 6.0 (yGPVR454 L-DOPA in HCl). FIG. 20B shows the levels of dopamine for the same culture over a 136 hour period. FIG. 20C shows the levels of reticuline for the same culture over a 136 hour period. FIG. 20D shows the levels of salutaridine for the same culture over a 136 hour period.

[00040] FIGs. 21A to 21F. FIG. 21A shows the pH of the media present when fermenting strain yGPVR454 over a 115 hour period. pH was regulated to keep pH above 6.0. Four test groups were used with repeats: 1) pH profile: pH never below 6.0 mostly at around 7.0 (similar to EF0269); 2) pH at 6.0 and transitioned to pH 6.5 after log72; 3) pH at 6.0; and 4) p02 20% and transitioned to 7% after log72. FIG. 21B shows the levels of dopamine for the same culture over a 115 hour period. FIG. 21C shows the levels of total reticuline for the same culture over a 115 hour period. FIG. 21D shows the levels of reticuline S for the same culture over a 115 hour period. FIG. 2 IE shows the levels of reticuline R for the same culture over a 115 hour period. FIG. 21F shows the levels of salutaridine for the same culture over a 115 hour period.

DETAILED DESCRIPTION OF THE DISCLOSURE

[00041] The following description and examples illustrate embodiments of the invention in

detail. It is to be understood that this invention is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this invention, which are encompassed within its scope.

[00042] BIAs can be produced in cells (e.g. , microorganisms) by genetic engineering. For

example, when producing morphinan alkaloids from microbial fermentation, a carbon substrate, such as sugar can be used to produce morphinan alkaloids.

[00043] In the first conversion, sugar, such as glucose, can be converted into L-tyrosine. FIG.

1A shows the molecular pathway from glucose to L-tyrosine in yeast. In bacteria, glucose can be converted into L-tyrosine by using a variety of enzymes. FIG. IB shows the molecular pathway from glucose to L-tyrosine in bacteria such as E. coli.

[00044] The second conversion, L-tyrosine to 1-DOPA can be performed by using a tyrosine

hydroxylase (e.g. , a cytochrome p450) to catalyze this reaction. The third conversion 1-DOPA to dopamine can be catalyzed by a DOPA decarboxylase (DODC). The fourth conversion makes use of one norcoclaurine synthase (NCS). The fifth conversion from (<S)-norcoclaurine to (S)/(R)-reticuline takes advantage of one or more of several enzymes including but not limited to: 60MT (6-<3-Methyltransferase), CNMT (coclaurine N-methyltransferase), NMCH

(cytochrome P450 N-methylcoclaurine hydroxylase a. lea. CYP80B1), 40MT (4-0- Methyltransferase) and REPI (reticuline epimerase). The sixth conversion of (R)-reticuline to salutaridine requires CPR (cytochrome P450 reductase) and SalSyn (salutaridine synthase). The seventh conversion of salutaridine to salutaridinol uses SalR (salutaridine reductase). The eighth conversion of salutaridinol to salutaridinol-7-O-acetate takes advantage of SalAT

(salutaridinol-7-O-acetyltransferase). The ninth conversion of salutaridmol-7-O-acetate to thebaine was previously thought to be a spontaneous process (e.g., at an elevated pH). However, it's been recently discovered that a thebaine synthase can catalyze this reaction, which is at least an order of magnitude more efficient compared to a spontaneous reaction.

[00045] The reactions to synthesize thebaine and some of the intermediates discussed above can be optimized to increase BIA production titers (or any of the intermediates described above). Control of the pH levels throughout the fermentation process can significantly increase BIA production titers. In some cases, steps can be taken to buffer the fermentation media during the fermentation process to optimal pH levels. This allows for the maintenance of a desired pH level in the growth/fermentation medium. During the fermentation process, the pH can be adjusted as needed, depending on the stages of the fermentation process.

[00046] Once the reaction proceeds to thebaine, the thebaine can be converted into derivative morphinan alkaloids such as oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxy codeinone, neopinone, and/or buprenorphine. For example, the conversion of thebaine to oripavine uses codeine O- demethylase (CODM). Oripavine can be further converted to morphinone using a thebaine 6-0- demethylase (T60DM). Morphinone can be converted to morphine using codeinone reductase (COR). COR can also convert morphine into morphinone. Thebaine can be converted into neopinone by a thebaine 6-O-demethylase. The reaction of neopinone into codeinone is believed to be spontaneous. Codeinone can be converted into codeine through the use of COR. The reverse reaction from codeine to codeinone can also be catalyzed by COR. Codeine can be converted into morphine by using a CODM.

[00047] Described herein are genetically modified microorganisms, enzymes, and methods to more efficiently produce BIAs, including thebaine, and other intermediates, from sugar.

DEFINITIONS

[00048] The term "about" in relation to a reference numerical value and its grammatical

equivalents as used herein can include the numerical value itself and a range of values plus or minus 10% from that numerical value. For example, the amount "about 10" includes 10 and any amounts from 9 to 1 1. For example, the term "about" in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. In some cases, the numerical disclosed throughout can be "about" that numerical value even without specifically mentioning the term "about."

[00049] The term "genetic modification " or "genetically modified" and their grammatical

equivalents as used herein can refer to one or more alterations of a nucleic acid, e.g., the nucleic acid within a microorganism's genome. For example, genetic modification can refer to alterations, additions, and/or deletion of nucleic acid (e.g., whole genes or fragments of genes).

[00050] The term "disrupting " and its grammatical equivalents as used herein can refer to a process of altering a gene, e.g., by deletion, insertion, mutation, rearrangement, or any combination thereof. For example, a gene can be disrupted by knockout or mutation.

Disrupting a gene can be partially reducing or completely suppressing expression (e.g., mRNA and/or protein expression) of the gene. Disrupting can also include inhibitory technology, such as shRNA, siRNA, microRNA, dominant negative, CRISPRi or any other means to inhibit functionality or expression of a gene or protein.

[00051] The term "gene editing " and its grammatical equivalents as used herein can refer to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome. For example, gene editing can be performed using a nuclease (e.g., a natural-existing nuclease or an artificially engineered nuclease).

[00052] The terms "and/or" and "any combination thereof and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases "A, B, and/or C" or "A, B, C, or any combination thereof can mean "A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C." [00053] The term "substantially pure" and its grammatical equivalents as used herein can mean that a particular substance does not contain a majority of another substance. For example, "substantially pure thebaine" can mean at least 90% thebaine. In some instances, "substantially pure thebaine" can mean at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 99.999%, or 99.9999% thebaine. For example, substantially pure thebaine can mean at least 70% thebaine. In some cases, substantially pure thebaine can mean at least 75% thebaine. In some cases, substantially pure thebaine can mean at least 80% thebaine. In some cases, substantially pure thebaine can mean at least 85% thebaine. In some cases, substantially pure thebaine can mean at least 90% thebaine. In some cases, substantially pure thebaine can mean at least 91% thebaine. In some cases, substantially pure thebaine can mean at least 92% thebaine. In some cases, substantially pure thebaine can mean at least 93% thebaine. In some cases, substantially pure thebaine can mean at least 94% thebaine. In some cases, substantially pure thebaine can mean at least 95% thebaine. In some cases, substantially pure thebaine can mean at least 96% thebaine. In some cases, substantially pure thebaine can mean at least 97% thebaine. In some cases, substantially pure thebaine can mean at least 98% thebaine. In some cases, substantially pure thebaine can mean at least 99% thebaine.

[00054] The term "heterologous" and its grammatical equivalents as used herein can mean

"derived from a different species." For example, a "heterologous gene" can mean a gene that is from a different species. In some instances, as "a yeast comprising a heterologous gene" can mean that the yeast contains a gene that is not from the same yeast. The gene can be from a different organism such as bacterium or plant or from a different species such as a different yeast species.

[00055] The term "substantially identical" and its grammatical equivalents in reference to another sequence as used herein can mean at least 50% identical. In some instances, the term substantially identical refers to a sequence that is 55% identical. In some instances, the term substantially identical refers to a sequence that is 60% identical. In some instances, the term substantially identical refers to a sequence that is 65% identical. In some instances, the term substantially identical refers to a sequence that is 70% identical. In some instances, the term substantially identical refers to a sequence that is 75% identical. In some instances, the term substantially identical refers to a sequence that is 80% identical. In other instances, the term substantially identical refers to a sequence that is 81% identical. In other instances, the term substantially identical refers to a sequence that is 82% identical. In other instances, the term substant lally identical refers to a sequence that is 83% identical. In other instances, the term substant ially identical refers to a sequence that is 84% identical. In other instances, the term substant ially identical refers to a sequence that is 85% identical. In other instances, the term substant ially identical refers to a sequence that is 86% identical. In other instances, the term substant ially identical refers to a sequence that is 87% identical. In other instances, the term substant ially identical refers to a sequence that is 88% identical. In other instances, the term substant ially identical refers to a sequence that is 89% identical. In some instances, the term substant ially identical refers to a sequence that is 90% identical. In some instances, the term substant ially identical refers to a sequence that is 91% identical. In some instances, the term substant ially identical refers to a sequence that is 92% identical. In some instances, the term substant ially identical refers to a sequence that is 93% identical. In some instances, the term substant ially identical refers to a sequence that is 94% identical. In some instances, the term substant ially identical refers to a sequence that is 95% identical. In some instances, the term substant ially identical refers to a sequence that is 96% identical. In some instances, the term substant ially identical refers to a sequence that is 97% identical. In some instances, the term substant ially identical refers to a sequence that is 98% identical. In some instances, the term substant ially identical refers to a sequence that is 99% identical. In order to determine the percentage of identity between two sequences, the two sequences are aligned, using for example the alignment method of Needleman and Wunsch (J. Mol. Biol, 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids/nucleotides is determined between the two sequences. For example, methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48: 1073) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs will be employed for such calculations. Computer programs that may be used in this regard include, but are not limited to, GCG (Devereux et al, Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et al, J. Molec. Biol., 1990:215:403). A particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994, Nucleic Acid Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.

[00056] The term "thebaine synthase", and its grammatical equivalents as used herein can refer to any polypeptide that can facilitate the conversion of a substrate into thebaine. For example, a thebaine synthase can be polypeptide that can convert salutaridinol-7-O-acetate into thebaine. In some cases, thebaine synthase can be called "Bet vl", "Bet-vl", "BETV1" (or derivatives, fragments, variants thereof) and the like.

[00057] The terms "salutaridinol 7-O-acetyltransferase", "Sal AT", and "SalAT polypeptide", and their grammatical equivalents as used herein can be used interchangeably, and can refer to any polypeptide that can facilitate the conversion of a substrate into salutaridinol 7-O-acetate. For example, SalAT can refer to any and all polypeptides that can convert salutaridinol to salutaridinol-7-O-acetate.

[00058] The terms "salutaridine reductase", "SalR", and "SalR polypeptide", and their

grammatical equivalents as used herein can be used interchangeably, and can refer to any polypeptide that can facilitate the conversion of a substrate into salutaridinol. For example, SalR can refer to any and all polypeptides that can convert salutaridine to salutaridinol.

GENERAL

[00059] Certain alkaloids belong to a class of chemical compounds known as benzylisoquinoline alkaloids ("BIAs"). Certain polypeptides are capable of mediating chemical reactions involving the conversion of a substrate (e.g., a carbon source) into a product (e.g., a BIA). Accordingly, disclosed are certain polypeptides capable of mediating chemical reactions involving conversion of a substrate into a BIA. Further, disclosed are methods that are extremely efficient at increasing salutaridine, salutaridinol, salutarindinol-7-O-acetate, and/or thebaine titers.

MICRORGANISMS USED IN THE SYNTHESIS OF BIAs

Cell-Types

[00060] The cells that can be used include but are not limited to plant or animal cells, fungus, yeast, algae, or bacterium. The cells can be prokaryotes or in some cases can be eukaryotes. For example, the cell can be aPapaver somniferum cell, Saccharomyces cerevisiae, Yarrowia lipolytica, or Escherichia coli, or any other cell disclosed throughout.

[00061] In certain cases, the cells are not naturally capable of producing BIAs (e.g. , thebaine or other morphinan alkaloids). In some cases, the cells are able to produce BIAs but at a low level. By implementation of the methods described herein, the cells can be modified such that the level of BIAs produced is higher relative to the level of the same BIA produced by the unmodified cells.

[00062] In some cases, the modified cell is capable of producing a substrate capable of being converted into a BIA, however, the cells are not capable of naturally producing a BIAs. The genetically modified microorganisms in some cases are unable to produce a substrate capable of being converted into a BIA, and the substrate capable of being converted into a BIA is provided to the cells as part of the cell's growth medium. In this case, the genetically modified microorganism can process the substrate capable of being converted into a BIA into a desired product such as thebaine or other BIA.

[00063] The cell can naturally comprise one or more enzyme capable of catalyzing one or more of the reactions: a sugar (or other carbon source capable of being converted into L-tyrosine such as glycerol and ethanol) to L-tyrosine; L-tyrosine to L-DOPA; L-DOPA to Dopamine;

Dopamine and 4-hydroxyphenylacetaldehyde to (<S)-Norcoclaurine (or norlaudanosoline); (S)- Norcoclaurine to (S)/(R)-Reticuline (in some cases, a subsequent conversion of (S)-reticuline to (R)-reticuline); (R)-Reticuline to Salutaridine; Salutaridine to Salutaridinol; or Salutaridinol to Salutaridinol-7-O-acetate.

Enzymes

[00064] The cells disclosed can be genetically modified with one or more enzymes that are capable of producing a BIA, such as thebaine, and other pathway intermediates such as reticuline, salutaridine and salutaridinol. The cell can be modified to include an enzyme that can perform any one of the following reactions: i) sugar (or other carbon source capable of being converted into L-tyrosine such as glycerol and ethanol) to l-tyrosine; ii) l-tyrosine to 1-DOPA; iii) 1-DOPA to dopamine; iv) dopamine and 4-hydroxyphenlacetaldehyde to (S)-norcoclaurine (or norlaudanosoline); v) (S)-norcoclaurine to (S)/(R)-reticuline (in some cases, a subsequent conversion of (S)-reticuline to (R)-reticuline); vi) (R)-reticuline to salutaridine; vii) salutaridine to salutaridinol; viii) salutaridinol to salutaridinol-7-O-acetate; ix) salutaridinol-7-O-acetate to the BIA thebaine; x) thebaine to other BIAs (such as oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxy codeinone, neopinone, and/or buprenorphme). For example, the cell can be modified with one or more of the following enzymes: tyrosine hydroxylase (TYR); DOPA decarboxylase (DODC);

norcoclaurine synthase (NCS); 6-O-Methyltransferase (60MT); coclaurine N-methyltransferase (CNMT), cytochrome P450 N-methylcoclaurine hydroxylase (NMCH), and 4-0- methyltransferase (40MT); cytochrome P450 reductase (CPR), salutaridine synthase (SalSyn); salutaridine reductase (SalR); salutaridinol-7-6>-acetyltransferase (SalAT); or any combination thereof. These enzymes can either be endogenous to the cell or heterologous. However, in some cases, even if the enzyme is endogenous, it can be made to be overexpressed. The heterologous enzymes can also be overexpressed.

[00065] In some cases, the SalAT polypeptide can be encoded by an amino acid sequence which is substantially identical to SEQ ID NO. 2. In some cases, the SalR polypeptide can comprise an amino acid sequence which is substantially identical to SEQ ID NO. 4.

[00066] Additionally, other enzymes can be used to make different products. These enzymes can include a thebaine synthase; a codeine O-demethylase (CODM); a thebaine 6-O-demethylase

(T60DM); a codeinone reductase (COR); or any combination thereof.

[00067] Some of the methods herein involve the use of thebaine synthases. The thebaine

synthases disclosed and used throughout, can be a polypeptide that is capable of converting salutaridinol-7-O-acetate to thebaine.

[00068] In some cases, the thebaine synthase can also be a polypeptide having an amino acid sequence that is substantially identical to any one of SEQ ID NOs. 6 to 8 and 43 to 57. For example, the thebaine synthase can be an amino acid sequence that is at least 50%, 60%, 70%,

80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs. 6 to 8 and

43 to 57.

[00069] In some cases, polypeptide that can catalyze the reaction of salutaridinol 7-O-acetate to thebaine can have a V _max of 4.0 nmol min ^"1 μg ^"1 . In some cases, the Vmax can be from 1.0 to 4.0 nmol min ^"1 μg ^"1 . In some cases, the ν _βχ can be from 1.5 to 3.5 nmol min ^"1 μg ^"1 . In some cases, the Vmax can be from 2.0 to 3.5 nmol min ^"1 μg ^"1 . In some cases, the Vmax can be from 2.5 to 4.0 nmol min ^"1 μg ^"1 .

[00070] In some cases, polypeptide that can catalyze the reaction of salutaridinol 7-O-acetate to thebaine can have a positive cooperativity ("η") of 2.3. In some cases, the positive cooperativity of the polypeptide can be from 2.0 to 2.5. In some cases, the positive cooperativity of the polypeptide can be from 2.1 to 2.4. In some cases, the positive cooperativity of the polypeptide can be from 2.2 to 2.3.

[00071] In some cases, the polypeptide that can catalyze the reaction of salutaridinol 7-O-acetate to thebaine can have a pH optimum of 8.0. In some cases, the pH optimum of the polypeptide can be from 6.5 to 9.0. In some cases, the pH optimum of the polypeptide can be from 7.0 to 8.5. In some cases, the pH optimum of the polypeptide can be from 7.5 to 8.0. [00072] In some cases, the polypeptide that can catalyze the reaction of salutaridinol 7-O-acetate to thebaine can do so using an S _N 2 mechanism. In some cases, the reaction can be supplemented with a reaction of (7S)-salutaridinol to thebaine using an S 2 mechanism, as described in FIG. IE. In some cases, the reaction can be supplemented with a reaction of (7S)-salutaridinol-7-0 acetate to thebaine using an S 2 mechanism, as described in FIG. 2E.

[00073] In some cases, a purine permease can be used in order to increase the productivity of the microorganism, e.g., increasing thebaine titers. The purine permease can have an amino acid sequence that is substantially identical to any one of SEQ ID NOs. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or 42.

[00074] In some cases, a cytochrome p450 reductase (CPR) can be used in order to increase the productivity of the microorganism, e.g., increasing thebaine titers. The cytochrome p450 reductase can have an amino acid sequence that is substantially identical to any one of SEQ ID NOs. 16, 67, 69, or 71.

[00075] The various combinations of enzymes can be used to make a desired product such as reticuline, salutandine, salutaridinol, and thebaine.

[00076] The enzymes disclosed throughout can be from a plant. For example, the enzymes can be from a plant that is from the genus Papaver. More specifically, Papaver plants that can be used include, but are not limited to Papaver bracteatum, Papaver somniferum, Papaver cylindricum, Papaver decaisnei, Papaver fugax, Papaver nudicale, Papaver oreophyllum, Papaver orientale, Papaver paeonifolium, Papaver persicum, Papaver pseudo-orientale, Papaver rhoeas, Papaver rhopalothece, Papaver armeniacum, Papaver setigerum, Papaver tauricolum, Papaver triniaefolium, Papaver rupifragium, Papaver apokrinomenon, Papaver spicatum, Papaver glaucum, Papaver burseri, Papaver alpinum, Papaver miyabeanum, Papaver lasiothrix, Papaver atlanticum, and Papaver radicatum. Papaver somniferum enzymes are particularly useful.

[00077] Additional enzymes can be added in order to improve the production of thebaine or other intermediates such as reticuline, salutaridine, salutaridinol, and salutaridinol-7-O-acetate.

Vectors

[00078] Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may typically, but not always, comprise a replication system (i.e. vector) recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and can preferably, but not necessarily, also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression systems (such as expression vectors) can include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome -binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, rriRNA stabilizing sequences, nucleotide sequences homologous to host chromosomal DNA, and/or a multiple cloning site. Signal peptides may also be included where appropriate, preferably from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.

[00079] The vectors can be constructed using standard methods (see, e.g., Sambrook et al, Molecular Biology: A Laboratory Manual, Cold Spring Harbor, N.Y. 1989; and Ausubel, et al , Current Protocols in Molecular Biology, Greene Publishing, Co. N.Y, 1995).

[00080] The manipulation of polynucleotides that encode the enzymes disclosed herein is

typically carried out in recombinant vectors. Numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes, episomal vectors and gene expression vectors, which can all be employed. A vector may be selected to accommodate a polynucleotide encoding a protein of a desired size. Following recombinant modification of a selected vector, a suitable host cell (e.g., the microorganisms described herein) is transfected or transformed with the vector. Each vector contains various functional components, which generally include a cloning site, an origin of replication and at least one selectable marker gene. A vector may additionally possess one or more of the following elements: an enhancer, promoter, and transcription termination and/or other signal sequences. Such sequence elements may be optimized for the selected host species. Such sequence elements may be positioned in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a preselected enzyme.

[00081] Vectors, including cloning and expression vectors, may contain nucleic acid sequences that enable the vector to replicate in one or more selected microorganisms. For example, the sequence may be one that enables the vector to replicate independently of the host chromosomal DNA and may include origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. For example, the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micron plasmid origin or CEN ARS are suitable for yeast, and various viral origins (e.g.SV40, adenovirus) are useful for cloning vectors. [00082] A cloning or expression vector may contain a selection gene (also referred to as a selectable marker). This gene encodes a protein necessary for the survival or growth of transformed microorganisms in a selective culture medium. Microorganisms not transformed with the vector containing the selection gene will therefore not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, G418, methotrexate, hygromycin, thiostrepton, apramycin or tetracycline, phleomycin, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.

[00083] The replication of vectors may be performed in E. coli. An E. co/z ^' -selectable marker, for example, the β-lactamase gene that confers resistance to the antibiotic ampicillin, may be of use. These selectable markers can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19, or pUC119.

Promoters

[00084] Vectors may contain a promoter that is recognized by the host microorganism. The

promoter may be operably linked to a coding sequence of interest. Such a promoter may be inducible or constitutive. Polynucleotides are operably linked when the polynucleotides are in a relationship permitting them to function in their intended manner.

[00085] Different promoters can be used to drive the expression of the genes. For example, if temporary gene expression (i.e., non-constitutively expressed) is desired, expression can be driven by inducible promoters.

[00086] In some cases, some of the genes disclosed can be expressed temporarily. In other

words, the genes are not constitutively expressed. The expression of the genes can be driven by inducible or repressible promoters. For example, the inducible or repressible promoters that can be used include but are not limited to: (a) sugars such as arabinose and lactose (or non metabolizable analogs, e.g., isopropyl β-D-l-thiogalactopyranoside (IPTG)); (b) metals such as lanthanum (or other rare earth metals such a cerium), copper, calcium; (c) temperature; (d) Nitrogen-source; (e) oxygen; (f) cell state (growth or stationary); (g) metabolites such as phosphate; (h) CRISPRi; (i) jun; (j) fos, (k) metallothionein and/or (1) heat shock.

[00087] Constitutively expressed promoters can also be used in the vector systems herein. For example, the expression of some of the genes disclosed throughout can be controlled by constitutively active promoters. For examples, the promoters that can be used include but are not limited to pGALl, pTEAl, pPGKl, pENOl, p.Bba.J23111, and J23100. [00088] Promoters suitable for use with prokaryotic hosts may include, for example, the a- lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, the erythromycin promoter, apramycin promoter, hygromycin promoter,

methylenomycin promoter and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Dalgarno sequence operably linked to the coding sequence.

[00089] Generally, a strong promoter may be employed to provide for high level transcription and expression of the desired product.

[00090] One or more promoters of a transcription unit can be an inducible promoter. For

example, a GFP can be expressed from a constitutive promoter while an inducible promoter drives transcription of a gene coding for one or more enzymes as disclosed herein and/or the amplifiable selectable marker.

[00091] Some vectors may contain prokaryotic sequences that facilitate the propagation of the vector in bacteria. Thus, the vectors may have other components such as an origin of replication (e.g., a nucleic acid sequence that enables the vector to replicate in one or more selected microorganisms), antibiotic resistance genes for selection in bacteria, and/or an amber stop codon which can permit translation to read through the codon. Additional selectable gene(s) may also be incorporated. Generally, in cloning vectors the origin of replication is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences can include the ColEl origin of replication in bacteria or other known sequences.

Genes

[00092] The genetically modified microorganisms can comprise a nucleic acid sequence

encoding for one or more enzymes that are capable of catalyzing one or more of the following reactions: i) sugar (or other carbon source capable of being converted into L-tyrosine such as glycerol and ethanol) to L-tyrosine; ii) L-tyrosine to L-DOPA; iii) L-DOPA to dopamine; iv) dopamine and 4-hydroxyphenylacetaldehyde to (<S)-norcoclaurine (or norlaudanosoline); v) (5)- norcoclaurine to (S)/(R)-reticuline (in some cases, a subsequent conversion of (S)-reticuline to (R)-reticuline); vi) (R)-reticuline to salutaridine; vii) salutaridine to salutaridinol; viii) salutaridinol to salutaridinol-7-O-acetate; ix) salutaridinol-7-O-acetate to the BIA thebaine; x) thebaine to other BIAs (such as oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxy codeinone, neopinone, and/or buprenorphine). For example, the genetically modified microorganism can comprise a nucleic acid sequence encoding for one or more of the following enzymes: tyrosine hydroxylase (TYR); DOPA decarboxylase (DODC); norcoclaunne synthase (NCS); 6-O-Methyltransferase (60MT); coclaurine N-methyltransferase (CNMT), cytochrome P450 N-methylcoclaurine hydroxylase (NMCH), and 4-< -methyltransferase (40MT); cytochrome P450 reductase (CPR), purine permease (PUP); salutaridine synthase (SalSyn); salutaridine reductase (SalR); salutaridinol-7- 0-acetyltransferase (SalAT); or any combination thereof. The nucleic acid sequence in some cases can be within a vector. In some cases, the nucleic acid sequences do not need to be within a vector but rather integrated into the microorganism's genome. In some cases, the isolated nucleic acid is inserted into the genome at a specific locus, where the isolated nucleic acid can be expressed in sufficient amounts.

[00093] In cases where a thebaine synthase is used, the thebaine synthase can be a polypeptide that is encoded by a polynucleotide that is substantially identical to SEQ ID NO. 5. For example, the thebaine synthase can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 5. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

[00094] The thebaine synthase can be encoded by a nucleic acid where the thebaine synthase has an amino acid sequence that is substantially identical to any one of SEQ ID NOs. 6 to 8 and 43 to 57. For example, the thebaine synthase can be encoded by a nucleic acid that encodes an amino acid sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO. 6 to 8 and 43 to 57.

[00095] In some cases, the thebaine synthase can be a fragment thereof. The fragment can still retain thebaine synthesis activity. In some cases, the activity of the thebaine synthase fragment can be decreased or increased compared to the activity produced by an polypeptide encoded by an amino acid sequence that is substantially identical to any one of SEQ ID NOs. 6 to 8 and 43 to 57. In one case, the thebaine synthase fragment can be encoded by a nucleic acid where the translated polypeptide has an amino acid sequence that is substantially identical to any one of SEQ ID NO. 6 to 8 and 43 to 57.

[00096] In certain instances, the SalAT polypeptide can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO. 1 or a fragment thereof. The SalR polypeptide can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO. 3 or a fragment thereof. [00097] In some cases, the CPR can be encoded by a polynucleotide that is substantially identical to any one of SEQ ID NOs. 15, 66, 68, or 70, or a fragment thereof.

[00098] In some cases, the PUP can be encoded by a polynucleotide that is substantially identical to any one of SEQ ID NOs. 19, 21, 23, 25, 27, 29, 31 , 33, 35, 37, 39, or 41 , or a fragment thereof.

[00099] The genetically modified microorganism can also further comprises one or more nucleic acids encoding for an enzyme capable of catalyzing one or more of the reactions:

a) a sugar (or other carbon source capable of being converted into L-tyrosine such as glycerol and ethanol) to L-tyrosine;

b) L-tyrosine to L-DOPA;

c) L-DOPA to Dopamine;

d) Dopamine and 4-hydroxyphenylacetaldehyde to (S)-Norcoclaurine (or norlaudanosoline);

e) (<S)-Norcoclaurine to (S)/(R)-Reticulme (in some cases, a subsequent conversion of (S)-reticuline to (R)-reticuline);

f) (R)-Reticuline to Salutaridine;

g) Salutaridine to Salutaridinol;

h) Salutaridinol to Salutaridinol-7-O-acetate; and/or

i) thebaine to oripavine, codeine, morphine, oxycodone, hydrocodone,

oxymorphone, hydromorphone, naltrexone, naloxone, hydroxy codeinone, neopinone, buprenorphine, or any combination thereof.

[000100] The genetically modified microorganism can also further one or more nucleic acids encoding for enzymes (in some cases heterologous enzymes), including but not limited to a codeine O-demethylase (CODM); a thebaine 6-O-demethylase (T60DM); a codeinone reductase (COR); or any combination thereof.

[000101] The genetically modified microorganism can also comprise one or more enzymes that can that confer upon the genetically modified microorganism the ability to enhance the production of a BIA. For example, a purine permease can be used to enhance the level of BIA production. Additionally, pH can affect the activity of such purine permease.

[000102] In some cases, when a purine permease is used, the purine permease can be a polypeptide that is encoded by a polynucleotide that is substantially identical to any one of SEQ ID NOs. 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, or 41. For example, the purine permease can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs. 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or 41. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

[000103] The purine permease when used can be encoded by a nucleic acid where the purine permease has an amino acid sequence that is substantially identical to any one of SEQ ID NOs. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or 42. For example, the purine permease can be encoded by a nucleic acid that encodes an amino acid sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or 42.

Modifying endogenous gene expression

[000104] The genetically modified microorganisms disclosed herein can have their endogenous genes regulated. This can be useful, for example, when there is negative feedback to the expression of a desired polypeptide, such as a thebaine synthase. Modifying this negative regulator can lead to increased expression of a desired polypeptide.

[000105] Modifying the expression of endogenous genes may be achieved in a variety of ways.

For example, antisense or RNA interference approaches may be used to down-regulate expression of the polynucleotides of the present disclosure, e.g., as a further mechanism for modulating cellular phenotype. That is, antisense sequences of the polynucleotides of the present disclosure, or subsequences thereof, may be used to block expression of naturally occurring homologous polynucleotide sequences. In particular, constructs comprising a desired polypeptide coding sequence, including fragments thereof, in antisense orientation, or combinations of sense and antisense orientation, may be used to decrease or effectively eliminate the expression of the desired polypeptide in a cell or plant and obtain an improvement in shelf life as is described herein. Accordingly, this may be used to "knock-out" the desired polypeptide or homologous sequences thereof. A variety of sense and antisense technologies, e.g. , as set forth in Lichtenstein and Nellen (Antisense Technology: A Practical Approach IRL Press at Oxford University, Oxford, England, 1997), can be used. Sense or antisense polynucleotide can be introduced into a cell, where they are transcribed. Such polynucleotides can include both simple oligonucleotide sequences and catalytic sequences such as ribozymes.

[000106] Other methods for a reducing or eliminating expression {i.e. , a "knock-out" or

"knockdown") of a desired polypeptide in a transgenic cell or plant can be done by introduction of a construct which expresses an antisense of the desired polypeptide coding strand or fragment thereof. For antisense suppression, the desired polypeptide cDNA or fragment thereof is arranged in reverse orientation (with respect to the coding sequence) relative to the promoter sequence in the expression vector. Further, the introduced sequence need not always correspond to the full length cDNA or gene, and need not be identical to the cDNA or gene found in the cell or plant to be transformed.

[000107] Additionally, the antisense sequence need only be capable of hybridizing to the target gene or RNA of interest. Thus, where the introduced polynucleotide sequence is of shorter length, a higher degree of homology to the endogenous transcription factor sequence will be needed for effective antisense suppression. While antisense sequences of various lengths can be utilized, in some embodiments, the introduced antisense polynucleotide sequence in the vector is at least 10, 20, 30, 40, 50, 100 or more nucleotides in length in certain embodiments.

Transcription of an antisense construct as described results in the production of RNA molecules that comprise a sequence that is the reverse complement of the mRNA molecules transcribed from the endogenous gene to be repressed.

[000108] Other methods for a reducing or eliminating expression can be done by introduction of a construct that expresses siRNA that targets a desired polypeptide. In certain embodiments, siRNAs are short (20 to 24-bp) double- stranded RNA (dsRNA) with phosphorylated 5' ends and hydroxylated 3' ends with two overhanging nucleotides.

[000109] Other methods for a reducing or eliminating expression can be done by insertion

mutagenesis using the T-DNA of ^' Agrobacterium tumefaciens or a selection marker cassette or any other non- sense DNA fragments. After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in the thebaine synthase (or other desired polypeptide) gene. Plants containing one or more transgene insertion events at the desired gene can be crossed to generate homozygous plant for the mutation, as described in Koncz et al, (Methods in Arabidopsis Research; World Scientific, 1992).

[000110] Suppression of gene expression may also be achieved using a ribozyme. Ribozymes are RNA molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,543,508. Synthetic ribozyme sequences including antisense RNAs can be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that hybridize to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.

[000111] The nucleotide sequence of a microorganism described herein can be altered by

homologous recombination. For example, nucleotides (e.g., genes) can be inserted or deleted using homologous recombination techniques. In particular, DNA sequences flanking a target coding sequence are useful for modification methods using homologous recombination. For example, for creating a deletion by homologous recombination, flanking sequences that are homologous to the target locus are placed on either sides of a selectable marker gene to mediate homologous recombination whereby the marker gene replaces the target gene. Also partial target gene sequences and flanking sequences bounding a selectable marker gene may be used to mediate homologous recombination whereby the marker gene replaces a portion of the target gene. In addition, the selectable marker may be bounded by site-specific recombination sites, so that following expression of the corresponding site-specific recombinase, the resistance gene is excised from the target gene without reactivating the latter. The site-specific recombination leaves behind a recombination site which disrupts expression of the target gene encoded protein. The homologous recombination vector may be constructed to also leave a deletion in the target gene following excision of the selectable marker.

[000112] A cell or plant gene may also be modified by using the Cre-lox system (for example, as described in U.S. Pat. No. 5,658,772). A cellular or plant genome can be modified to include first and second lox sites that are then contacted with a Cre recombinase. If the lox sites are in the same orientation, the intervening DNA sequence between the two sites is excised. If the lox sites are in the opposite orientation, the intervening sequence is inverted.

[000113] In addition, silencing approach using short hairpin RNA (shRNA) system, and

complementary mature CRISPR RNA (crRNA) by CRISPR/Cas system, and virus inducing gene silencing (VIGS) system may also be used to make down regulated or knockout of synthase mutants. Dominant negative approaches may also be used to make down regulated or knockout of desired polypeptides.

[000114] The RNA-guided endonuclease can be derived from a clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. The CRISPR Cas system can be a type I, a type II, or a type III system. Non-limiting examples of suitable CRISPR/Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966.

[000115] In general, CRISPR/Cas proteins comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains interact with guide RNAs. CRISPR/Cas proteins can also comprise nuclease domains (i.e. , DNase or RNase domains), DNA binding domains, helicase domains, RNase domains, protein-protein interaction domains, dimerization domains, as well as other domains.

[000116] The CRISPR/Cas-like protein can be a wild type CRISPR/Cas protein, a modified

CRISPR/Cas protein, or a fragment of a wild type or modified CRISPR Cas protein. The CRISPR/Cas-like protein can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzyme activity, and/or change another property of the protein. For example, nuclease (i.e. , DNase, RNase) domains of the CRISPR/Cas-like protein can be modified, deleted, or inactivated. Alternatively, the CRISPR/Cas-like protein can be truncated to remove domains that are not essential for the function of the fusion protein. The CRISPR/Cas-like protein can also be truncated or modified to optimize the activity of the effector domain of the fusion protein.

[000117] One method to silence a desired gene is virus induced gene silencing (known to the art as VIGS). In general, in plants infected with unmodified viruses, the viral genome is targeted. However, when viral vectors have been modified to carry inserts derived from host genes (e.g. portions of sequences encoding a desired polypeptide) the process is additionally targeted against the corresponding mRNAs. Thus disclosed is a method of producing a plant expressing reduced levels of a desired gene or other desired gene(s), the method comprising (a) providing a plant expressing a desired gene; and (b) reducing expression of the desired gene in the plant using virus induced gene silencing.

Exemplary Genetically Modified Microorganisms

[000118] Disclosed herein is a genetically modified microorganism capable of converting a carbon substrate into a benzylisoquinoline alkaloid (BIA). In some instances, the genetically modified microorganism comprises a heterologous nucleic acid encoding a proton pump.

[000119] The genetically modified microorganism can further comprise a heterologous

polynucleotide encoding a cytochrome P450 reductase (CPR); purine permease (PUP);

salutaridine synthase (Sal Syn); salutaridine reductase (SalR); salutaridinol 7-O-acetyltransferase (SalAT); or thebaine synthase (THS). In some cases, two or more heterologous polynucleotides encoding Sal Syn, SalR, SalAT, or THS can be present within the genetically modified microorganism. In some cases, three or more heterologous polynucleotides encoding Sal Syn, SalR, SalAT, or THS can be present within the genetically modified microorganism. In some cases, all of the heterologous polynucleotides encoding Sal Syn, SalR, SalAT, or THS can be present within the genetically modified microorganism. [000120] Should a Sal Syn be present within the genetically modified microorganism, the Sal Syn can be encoded by an amino acid sequence substantially identical to SEQ ID NOs. 10 or 18. In some cases, the Sal Syn can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NOs. 9 or 17.

[000121] Further, should the genetically modified microorganism have a Sal R, the Sal R can be encoded by an amino acid sequence substantially identical to SEQ ID NO. 12. In some cases, the Sal R can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO. 11.

[000122] Should the genetically modified microorganism have a SalAT, the SalAT can be encoded by an amino acid sequence substantially identical to SEQ ID NO. 14. In some cases, the SalAT can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO. 13.

[000123] Should a THS be present within the genetically modified microorganism, the THS can be encoded by an amino acid sequence substantially identical to SEQ ID NO. 6. In some cases, the THS can be encoded by an amino acid sequence substantially identical to SEQ ID NO. 7. In some cases, the THS can be encoded by an amino acid sequence substantially identical to SEQ ID NO. 8. In some cases, the THS can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO. 5.

[000124] Additionally, these genetically modified microorganisms can further comprise a

cytochrome P450 reductase (CPR). The CPR can be encoded by an amino acid sequence substantially identical to any one of SEQ ID NOs. 16, 67, 69, or 71.

[000125] These genetically modified microorganisms can further comprise a purine permease (PUP). The PUP can be encoded by an amino acid sequence substantially identical to SEQ ID NOs. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or 42.

[000126] In certain cases, the genetically modified microorganism can be a yeast, algae, or

bacterium. Should the genetically modified microorganism be a yeast, the yeast can be from the genus Saccharomyces . More specifically, the yeast can be from the species Saccharomyces cerevisiae.

[000127] The genetically modified microorganism can use sugar, ethanol, glycerol, tyrosine, L- DOPA, or dopamine as a substrate. In some cases, the genetically modified microorganism can make a BIA, where the BIA is thebaine, morphine, codeine, onpavine, oxycodone, hydrocodone, or oxymorphone.

[000128] The genetically modified microorganism can also further comprise one or more nucleic acids encoding for an enzyme capable of catalyzing one or more of the reactions: i) a sugar (or other carbon source capable of being converted into L-tyrosme such as glycerol and ethanol) to L-tyrosine; ii) L-tyrosine to L-DOPA; iii) L-DOPA to Dopamine; iv) Dopamine and 4- hydroxyphenylacetaldehyde to (S)-Norcoclaurine (or norlaudanosoline); v) (S)-Norcoclaurine to (S)/( )-Reticuline; or vi) thebaine to oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxy codeinone, neopinone, buprenorphine, or any combination thereof.

FERMENTATION METHODS AND PROCESSES

[000129] In general, the microorganisms disclosed herein should be used in fermentation

conditions that are appropriate to convert a carbon source (such as a sugar, ethanol, or glycerol) to a BIA (thebaine, other morphinan alkaloids, or morphinan alkaloid derivatives). Reaction conditions that should be considered include temperature, media flow rate, pH, media redox potential, agitation rate, inoculum level, maximum substrate concentrations and rates of introduction of the substrate to the bioreactor to ensure that substrate level does not become limiting, and maximum product concentrations to avoid product inhibition.

[000130] In some cases, non-genetically modified microorganisms can be used to increase BIA production. For example, cells taken from organisms that naturally produce BIAs can be used. These cells can be isolated and once isolated they can be used in a fermentation process.

Fermentation Conditions

[000131] The fermentation conditions described herein are applicable to any and all methods disclosed throughout the application.

nK

[000132] pH can greatly alter the activity of one or more of the enzymes disclosed. Therefore, as fermentation progresses, pH can be optimized based on the type of enzymes used and the end product desired.

[000133] In some cases, the pH during fermentation can vary from 4 to 10. In some instances, the pH can be from 5 to 9; 6 to 8; 6.1 to 7.9; 6.2 to 7.8; 6.3 to 7.7; 6.4 to 7.6; or 6.5 to 7.5. For example, the pH can be from 6.6 to 7.4. In some instances, the pH can be from 5 to 9. In some instances, the pH can be from 6 to 8. In some instances, the pH can be from 6.1 to 7.9. In some instances, the pH can be from 6.2 to 7.8. In some mstances, the pH can be from 6.3 to 7.7. In some instances, the pH can be from 6.4 to 7.6. In some instances, the pH can be from 6.5 to 7.5. In some instances the pH used for the fermentation can be greater than 6. In some instances the pH used for the fermentation can be lower than 10. [000134] For example, in order to generate salutaridine, the pH of the medium containing the genetically modified microorganism (or the pH of the microorganism itself) should be optimized accordingly. As shown in FIG. 8, a genetically modified microorganism expressing SalSyn, SalR, SalAT, and CPR, produce the highest titers of salutaridine between pH 7.5 and 9.2.

Further, the same organism produced the highest titers of salutaridinol at a pH of between 5.5 and 8.5. Thebaine tiers produced by the genetically modified microorganism were the highest at a pH of between 5.5 and 8.

[000135] In some cases, the pH can be adjusted above a certain level. For example, when the pH of the fermentation media reaches a pH of 4.0, a base can be added to the fermentation media to increase the pH. In some cases, when the pH of the fermentation media reaches pH 5.0, a base is added to the fermentation media. In some cases, when the pH of the fermentation media reaches pH 5.5, a base is added to the fermentation media. In some cases, when the pH of the fermentation media reaches pH 6.0, a base is added to the fermentation media. In some cases, when the pH of the fermentation media reaches pH 6.5, a base is added to the fermentation media. In some cases, when the pH of the fermentation media reaches pH 7.0, a base is added to the fermentation media. In some cases, when the pH of the fermentation media reaches pH 7.5, a base is added to the fermentation media. In some cases, when the pH of the fermentation media reaches pH 8.0, a base is added to the fermentation media.

[000136] In some cases, the pH can be constantly held at a specific pH by adding an acid or base to the media. For example, the pH can be held constant at pH 6. In some cases, the pH can be held constant at pH 6.5. In some cases, the pH can be held constant at pH 7.0. In some cases, the pH can be held constant at pH 7.5. In some cases, the pH can be held constant at pH 8.0. In some cases, the pH can be held constant at pH 8.5. In some cases, the pH can be held constant at pH 9.0. In some cases, the pH can be held constant at pH 9.5. In some cases, the pH can be held constant at pH 10.0.

[000137] In some cases, the pH can be constantly held at a specific pH range by adding an acid or base to the media. For example, the pH can be held at a range of between pH 4 and 10. In some cases, the pH can be held at a range of pH 4.5 to 9.5. In some cases, the pH can be held at a range of pH 5.0 to 9.0. In some cases, the pH can be held at a range of pH 5.5 to 9.0. In some cases, the pH can be held at a range of pH 6.0 to 9.0. In some cases, the pH can be held at a range of pH 6.5 to 9.0. In some cases, the pH can be held at a range of pH 7.0 to 9.0. In some cases, the pH can be held at a range of pH 7.5 to 9.0. In some cases, the pH can be held at a range of pH 8.0 to 8.5. [000138] The timing of the pH adjustment can vary depending on the stage of fermentation. For example, pH adjustment may begin after the growth stage, when the microorganisms start to perform the fermentation process. In some cases, this can occur 24 to 48 hours after the initial inoculation of the media. In some cases, the pH adjustment can be before 24 hours or after 48 hours.

Temperature

[000139] Temperature can also be adjusted based on the microorganism used or enzyme

sensitivity. For example, the temperature used during fermentation, can from 25 C° to 45 C°. In other instances, the temperature of the fermentation can be from 22 C° to 40 C°; 24 C° to 39 C°; 25 C° to 38 C°; 26 C° to 37 C°; 28 C° to 40 C°; 30 C° to 45 C°; 31 C° to 44 C°; 32 C° to 43 C°; 33 C° to 42 C°; 34 C° to 41 C°; 35 C° to 40 C°. For example, the temperature can be from 36 C° to 39 C° (e.g., 36 C°, 37 C°, 38 C°, or 39 C°). In some instances, the temperature can be from 30 C° to 45 C°. In some instances, the temperature can be from 31 C° to 44 C°. In some instances, the temperature can be from 32 C° to 43 C°. In some instances, the temperature can be from 33 C° to 42 C°. In some instances, the temperature can be from 34 C° to 41 C°. In some instances, the temperature can be from 35 C° to 40 C°.

Gases

[000140] Availability of oxygen and other gases such as gaseous CO ₂ can affect yield and

fermentation rate. For example, when considering oxygen availability, the percent of dissolved oxygen (DO) within the fermentation media can be from 1% to 40%. In certain instances, the DO concentration can be from 1.5% to 35%; 2% to 30%; 2.5% to 25%; 3% to 20%; 4% to 19%; 5% to 18%; 6% to 17%; 7% to 16%; 8% to 15%; 9% to 14%; 10% to 13%; or 11% to 12%. For example, in some cases the DO concentration can be from 2% to 30%. In other cases, the DO can be from 3% to 20%. In some instances, the DO can be from 4% to 10%. In some cases, the DO can be from 1.5% to 35%. In some cases, the DO can be from 2.5% to 25%. In some cases, the DO can be from 4% to 19%. In some cases, the DO can be from 5% to 18%. In some cases, the DO can be from 6% to 17%. In some cases, the DO can be from 7% to 16%. In some cases, the DO can be from 8% to 15%. In some cases, the DO can be from 9% to 14%. In some cases, the DO can be from 10% to 13%. In some cases, the DO can be from 11% to 12%.

[000141] For example, when considering atmospheric CO ₂ , the percent of atmospheric CO ₂ within an incubator can be from 0% to 10%. In some cases, atmospheric CO ₂ can help to control the pH within cell culture medium. pH contain within cell culture media is dependent on a balance of dissolved CO ₂ and bicarbonate (HCO3). Changes in atmospheric CO ₂ can alter the pH of the medium. In certain instances, the atmospheric CO ₂ can be from 0% to 10%; 0.01% to 9%;

0.05% to 8%; 0.1% to 7%; 0.5% to 6%; 1% to 5%; 2% to 4%; 3% to 6%; 4% to 7%; 2% to 6%; or 5% to 10%. For example, in some cases the atmospheric CO ₂ can be from 0% to 10%. In other cases, the atmospheric CO ₂ can be from 0.01% to 9%. In some instances, the atmospheric CO ₂ can be from 0.05% to 8%. In some cases, the atmospheric CO ₂ can be from 0.1% to 7%. In some cases, the atmospheric CO ₂ can be from 0.5% to 6%. In some cases, the atmospheric CO ₂ can be from 1% to 5%. In some cases, the atmospheric CO ₂ can be from 2% to 4%. In some cases, the atmospheric CO ₂ can be from 3% to 6%. In some cases, the atmospheric CO ₂ can be from 4% to 7%. In some cases, the atmospheric CO ₂ can be from 2% to 6%. In some cases, the atmospheric CO ₂ can be from 5% to 10%.

Fermentation Time

[000142] The methods as disclosed throughout can be performed for specific lengths of time. In some cases, the reaction time can be vary.

[000143] In some cases, the reaction time can be less than 60 minutes. For example, in some cases, fermentation with one or more of the enzymes disclosed throughout can be between 1 second and 600 seconds (i.e., 10 minutes). In some cases, the fermentation time can be between 1 second and 540 seconds. In some cases, the fermentation time can be between 1 second and 480 seconds. In some cases, the fermentation time can be between 1 second and 420 seconds. In some cases, the fermentation time can be between 1 second and 360 seconds. In some cases, the fermentation time can be between 1 second and 300 seconds. In some cases, the fermentation time can be between 1 second and 240 seconds. In some cases, the fermentation time can be between 1 second and 180 seconds. In some cases, the fermentation time can be between 1 second and 120 seconds. In some cases, the fermentation time can be between 1 second and 90 seconds. In some cases, the fermentation time can be between 1 second and 60 seconds. In some cases, the fermentation time can be between 1 second and 45 seconds. In some cases, the fermentation time can be between 1 second and 30 seconds. In some cases, the fermentation time can be between 1 second and 15 seconds. In some cases, fermentation time can be about: 600 seconds, 540 seconds, 480 seconds, 420 seconds, 360 seconds, 300 seconds, 240 seconds, 180 seconds, 120 seconds, 60 seconds, 45 seconds, 30 seconds, or 15 seconds. In some cases, the fermentation time can be about 30 seconds. In some cases, the fermentation time can be about 60 seconds.

[000144] In some cases, fermentation time can be greater than 12 hours. For example, in some cases, fermentation with one or more enzymes disclosed throughout can be between 12 hours and 200 hours. In some cases, fermentation time can be between 12 hours and 180 hours. In some cases, fermentation time can be between 12 hours and 160 hours. In some cases, fermentation time can be between 12 hours and 144 hours. In some cases, fermentation time can be between 12 hours and 132 hours. In some cases, fermentation time can be between 12 hours and 120 hours. In some cases, fermentation time can be between 12 hours and 116 hours. In some cases, fermentation time can be between 12 hours and 112 hours. In some cases, fermentation time can be between 12 hours and 104 hours. In some cases, fermentation time can be between 12 hours and 100 hours. In some cases, fermentation time can be between 12 hours and 96 hours. In some cases, fermentation time can be between 12 hours and 88 hours. In some cases, fermentation time can be between 12 hours and 80 hours. In some cases, fermentation time can be between 12 hours and 72 hours. In some cases, fermentation time can be between 12 hours and 66 hours. In some cases, fermentation time can be between 12 hours and 60 hours. In some cases, fermentation time can be between 12 hours and 48 hours. In some cases, fermentation time can be between 24 hours and 48 hours. In some cases, fermentation time can be about: 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, 168 hours, 180 hours, 192 hours, 204 hours, or 126 hours.

Bioreactors

[000145] Fermentation reactions may be carried out in any suitable bioreactor. In some

embodiments of the invention, the bioreactor may comprise a first, growth reactor in which the micro-organisms are cultured, and a second, fermentation reactor, to which broth from the growth reactor is fed and in which most of the fermentation product (for example, thebaine or other BIAs) is produced.

Product Recovery

[000146] The fermentation of the microorganisms disclosed herein can produce a fermentation broth comprising a desired product {e.g., thebaine or other BIA) and/or one or more by-products as well as the microorganisms (e.g. , a genetically modified microorganism), in a nutrient medium.

[000147] In certain embodiments the thebaine or other BIA produced in the fermentation reaction is converted to morphine, codeine or other morphine derivatives or morphine like products, like oxycodone. This conversion can happen directly from the fermentation broth. However, in other embodiments, the thebaine or other BIA can be first recovered from the fermentation broth before conversion to morphine, codeine or other morphine derivatives or morphine-like products, such as oxycodone.

[000148] In some cases, the thebaine or other BIA can be continuously removed from a portion of broth and recovered as purified the thebaine or other BIA. In particular embodiments, the recovery of the thebaine or other BIA includes passing the removed portion of the broth containing the thebaine or other BIA through a separation unit to separate the microorganisms (e.g., genetically modified microorganism) from the broth, to produce a cell -free the thebaine or other BIA containing permeate, and returning the microorganisms to the bioreactor. Additional nutrients may be added to the media to replenish its nutrients before it is returned to the bioreactor. The cell-free the thebaine or other BIA-containing permeate may then can be stored or be used for subsequent conversion to morphine, codeine or other morphine derivatives or morphine-like products, such as oxycodone (or other desired product).

[000149] Also, if the pH of the broth was adjusted during recovery of thebaine, other BIAs,

morphine, codeine or other morphine derivatives or morphine-like products, like oxycodone, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.

[000150] Subsequent purification steps may involve treating the post-fermentation thebaine or other BIA product using methods known in the art to recover individual product species of interest to high purity.

[000151] In one example, thebaine or other BIAs (including BIA precursors) extracted in an

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

[000152] The thebaine or other BIAs containing solution may be further treated to remove metals, for example, by treating with a suitable chelating agent. The thebaine or other BIAs containing solution may be further treated to remove other impurities, such as proteins and DNA, by precipitation. In one example, the thebaine or other BIAs containing solution is treated with an appropriate precipitation agent such as ethanol, methanol, acetone, or isopropanol. In an alternative example, DNA and protein may be removed by dialysis or by other methods of size exclusion that separate the smaller alkaloids from contaminating biological macromolecules. [000153] In further examples, the thebaine or other BIAs-containing solution may be extracted to high purity by continuous cross-flow filtration using methods known in the art.

[000154] If the solution contains a mixture of thebaine or other BIAs, it may be subjected to acid- base treatment to yield individual BIAs of interest species using methods known in the art. In this process, the pH of the aqueous solution is adjusted to precipitate individual BIAs (such as thebaine or other BIAs) at their respective pKas.

[000155] For high purity, small-scale preparations, the thebaine or other BIAs may be purified in a single step by liquid chromatography.

Methods of making BIAs (e.g., thebaine)

[000156] The genetically modified microorganisms described throughout can be used to make BIAs, e.g., thebaine, morphine, and their derivatives. A substrate capable of being converted into a BIA can be brought in contact with a thebaine synthase in a reaction mixture under reaction conditions permitting a thebaine synthase mediated reaction resulting in the conversion of the substrate into thebaine, or other BIA. Under such reaction conditions living cells are modified in such a manner that they produce BIAs, e.g. , thebaine or morphine and its derivatives.

[000157] The BIAs {e.g. , thebaine, morphine, and their derivatives) produced may be recovered and isolated from the modified cells. The BIAs (thebaine, morphine, and their derivatives) in some cases may be secreted into the media of a cell culture, in which the BIA is extracted directly from the media. In some cases, the BIA may be within the cell itself, and the cells will need to be lysed in order to recover the BIA. In some cases, the cells can be lysed and BIAs produced in a cell free or in vitro reaction. In some instances, both cases may be true, where some BIAs are secreted and some remains within the cells. In this case, either method or both methods can be used.

[000158] Accordingly, disclosed herein is a method of making a benzylisoquinoline alkaloid

(BIA) comprising (a) contacting the genetically modified microorganism with a medium comprising a carbon source, and (b) growing the genetically modified microorganism to produce said BIA. The genetically modified microorganism can comprise any microorganism disclosed throughout. For example, the microorganism can be a genetically modified microorganism capable of converting a carbon substrate into a benzylisoquinoline alkaloid (BIA), said genetically modified microorganism comprising a heterologous nucleic acid encoding a proton pump. [000159] Also disclosed is a method of making salutaridine comprising contacting Sal Syn with reticuline within a medium to convert reticuline to salutaridine, wherein the pH of said medium is between 6 and 13. For example, in some cases, the pH of said media can be between 6 and 8.

[000160] Also disclosed is a method of making salutaridinol comprising contacting SalR with salutaridine within a medium to convert salutaridine to salutaridinol, wherein the pH of said medium is between 5 and 13.

[000161] Further disclosed is a method of making salutaridinol-7-O-acetate comprising contacting SalAT with salutaridinol within a medium to convert salutaridinol to salutaridinol -7-0 -acetate, wherein the pH of said medium is between 6 and 13.

[000162] Additionally disclosed is a method of making thebaine comprising contacting THS with to salutaridinol-7-O-acetate within a medium to convert to salutaridinol-7-O-acetate to thebaine, wherein the pH of said medium is between 5 and 13.

[000163] In some cases, for example, when the methods include a microorganism (in other words, when the process is done in an in vivo setting rather than an in vitro setting), the microorganism can include a PUP or a CPR. If a PUP is used, the pH of the media can be adjusted to a pH of between 6.0 to 8.0 (e.g. , 6.0 to 8.0; 6.5 to 7.7.0; 7.0 to 7.6; or 7.3 to 7.5), or otherwise disclosed throughout the application. If a CPR is used, the pH of the media can be adjusted to a pH of between 5.0 to 8.0 (e.g. , 5.5 to 8.0; 6.0 to 7.7; 6.5 to 7.5; or 7.0 to 7.4), or otherwise disclosed throughout the application.

[000164] In some cases, when the methods include only a cell-free system (or a system without live cells) the pH used can be different. For example, if a PUP is used, the pH of the media can be adjusted to a pH of between 6.0 to 10.0 (e.g., 6.0 to 9.5; 6.5 to 9.0; 7.0 to 8.5; or 7.5 to 8.0), or otherwise disclosed throughout the application. If a CPR is used, the pH of the media can be adjusted to a pH of between 5.0 to 10.0 (e.g., 5.5 to 9.5; 6.0 to 9.0; 6.5 to 8.5; or 7.0 to 8.0), or otherwise disclosed throughout the application.

[000165] The pH of the medium can be adjusted during the process of growing the genetically modified microorganism. For example, the pH can be adjusted to any of the pHs disclosed throughout. In some case, the pH can be adjusted to greater than 6. In some cases, the pH is adjusted to between 6 to 6.4. In some cases, the pH is adjusted to between 6.5 to 6.9. In some cases, the pH is adjusted to between 7 to 7.4. In some instances, the pH is adjusted to between 7.5 to 7.9. In other cases, the pH is adjusted to between 8 to 8.4. In some instances, the pH can be adjusted by supplementing said medium with an acidic or alkali substance. For example, if the medium is acidic (i.e., under a pH of 7), then an alkali substance can be used to buffer the media. Some alkali substances or buffering reagents that can be used include but are not limited to sodium hydroxide (NaOH), ammonium hydroxide (NH ₄ OH), potassium hydroxide (KOH), 4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES buffer), 2-(N- moφholino)ethanesulfonic acid (MES buffer). Some acidic substances that can be used include but are not limited to hydrochloric acid (HCl), acetate, sulfuric acid (H ₂ SO ₄ ) or any combination thereof.

[000166] The carbon source can be any carbon source that can be used by the microorganism. In some cases, the carbon source can be a sugar, such as glucose, fructose, galactose, mannose, or any combination thereof. In some cases, the carbon source can be oleate, glycerol, acetate or ethanol, L-tyrosine, tyramine, L-3,4-dihydroxyphenyl alanine (L-DOPA), dopamine, or any combination thereof.

[000167] The BIA produced by the methods disclosed throughout can be any BIA including but not limited to thebaine. Other BIAs can include but are not limited to norcoclaurine, norlaudanosolme, reticulme, noscapine, morphine, dihydrocodeine, codeine, codeinone, morphinone, oripavine, oxycodone, hydrocodone, and oxymorphone, hydromorphone, noroxymorphone, nalonxone, and naltrexone.

[000168] In some cases, the medium does not contain any intact cells. In other words, this

reaction is performed in the media in vitro or a cell-free system. The reaction does not occur within the cell. For example, the conversion of reticuline to salutaridine can occur outside of a cell, e.g., in media or buffer containing lysed cells. In some cases, the conversion of salutaridine to salutaridinol can occurs outside of a cell. In some cases, the conversion of salutaridinol to salutaridinol-7-O-acetate can occur outside of a cell. In some cases, the cells are lysed and the cellular components are exposed to the media and the conversion occurs in the media. One or more the enzymes described throughout can perform its reaction in vitro to catalyze enzymatic reactions. In some cases, these enzymes can be native or recombinant to the cell, and isolated. At specific times, the enzymes can be added back to a reaction medium and the enzymes can perform its designed function. The reaction can be stopped by denaturing the enzyme (e.g., by heating up the reaction). In some cases, stopping the previous reaction is not necessary and the next enzyme can be added to the reaction media to perform a next step.

[000169] In some cases, this reaction is contained within a cell grown in cell culture media. In other words, the reaction is performed in vivo. For example, the conversion of reticuline to salutaridine can occur within a cell. In some cases, the conversion of salutaridine to salutaridinol can occurs within a cell. In some cases, the conversion of salutaridinol to salutaridinol-7-O-acetate can occur within a cell.

[000170] In some cases, the conversions can occur within the cell and travel to the outside of the cells. In some cases, the conversions can occur outside of the cell and travel into the cell.

[000171] In some cases, there is a combination of the two. Some reactions along the BIA pathway can occur within a cell, whereas some of the reactions along the BIA pathway occur outside of a cell.

[000172] In some cases, the medium is cell culture media. In other instances, the medium is water or other liquid in which the cells (for in vivo reactions) can survive (such as saline buffered water). In other instances, the medium is water or other liquid in which the enzymes (for in vitro reactions) are active.

[000173] Where reticuline is necessary, the reticuline can be S-reticuline or R-reticuline. However, in some cases, R-reticuline is required and is used. In some cases S-reticuline can be converted into R-reticuline by a REPI enzyme.

[000174] In general, a pH higher than 6.0 results in a more efficient conversion of a carbon into a BIA compared to a reaction that occurs at a lower pH, when everything else is equal. In this regard, a conversion is deemed "more efficient" if larger quantities of BIAs are obtained in the reaction mixture upon substantial completion of the reaction and/or if the BIA accumulates in the reaction mixture at a faster rate. However, in some cases, the pH of the media can be adjusted according to any method described throughout. In some case, the pH can be adjusted to greater than 5. In some instances, the pH is adjusted to between 5.5 to 5.9. In some case, the pH can be adjusted to greater than 6. In some instances, the pH is adjusted to between 6 to 6.4. In some case, the pH can be adjusted to greater than 6. In some instances, the pH is adjusted to between 6.5 to 6.9. In some cases, the pH is adjusted to between 7 to 7.4. In some instances, the pH is adjusted to between 7.5 to 7.9. In other cases, the pH is adjusted to between 8 to 8.4.

[000175] The timing of pH buffer can also affect the final output of BIAs. For example, after each reaction, the optimal pH needed for the subsequent enzyme may be different, and therefore may need to be adjusted according. For example, Sal Syn working optimally above a pH of 7.

However, SalR may work at a lower pH, e.g. , between 5.5 and 8.5. THS may work again optimally above a pH of 7. Therefore, at certain points, the reaction may need to be adjusted to a pH of higher than 7 (e.g., 7.5), then back to a pH of below 7 (e.g., 6), then once again back to above a pH of 7. [000176] The output of the methods disclosed throughout can be further converted into other targets. For example, if the method produces salutaridinol-7-O-acetate, the method can further comprise contacting that product (in this case salutaridinol-7-O-acetate) with an enzyme that is capable of converting it into another product (in this case THS). In this case, thebaine can be made should salutaridinol-7-O-acetate be contacted with THS. Any enzyme or combination of enzymes can be used to convert the product of the methods disclosed throughout into an upstream or downstream product. Some of the enzymes that can be used include those shown in FIG. 1C and FIG. 7A. Some of the products can include without limitation, oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, neopinone, hydroxycodeinone, buprenorphine, or any combination thereof.

[000177] The BIAs produced herein can be useful inter alia in the manufacture of pharmaceutical compositions. Thus, disclosed herein is a method of making a pharmaceutical composition by using the products disclosed herein.

[000178] Further, other additives can be included in a reaction mixture, such as cofactors like ATP, SAM, NADPH and acetyl-CoA. In particular, reaction mixtures comprising SalAT further comprise Acetyl-CoA, and reaction mixtures comprising SalR further comprise NADPH.

[000179] Upon completion of the methods or reactions described throughout, the amount of a

particular BIA, e.g. , thebaine or morphine, present in the reaction mixture can be at least 0.1% (w/w), at least 0.25% (w/w), at least 0.5% (w/w), at least 0.75% (w/w), at least 1.0% (w/w), at least, 1.5% (w/w), or at least 2.0% (w/w) of the total BIAs in the reaction mixture. In some instances, the reaction mixture comprises thebaine in a weight percentage of least 0.1% (w/w), at least 0.25% (w/w), at least 0.5% (w/w), at least 0.75% (w/w), at least 1.0% (w/w), at least, 1.5% (w/w), or at least 2.0% (w/w) of total BIAs in the reaction mixture. In some other cases, the reaction mixture comprises salutaridinol 7-O-acetate, in a weight percentage of at least 0.1% (w/w), at least 0.25% (w/w), at least 0.5% (w/w), at least 0.75% (w/w), at least 1.0% (w/w), at least, 1.5% (w/w), or at least 2.0% (w/w)of total BIAs in the reaction mixture. In some other cases, the reaction mixture comprises salutaridinol, in a weight percentage of at least 0.1% (w/w), at least 0.25% (w/w), at least 0.5% (w/w), at least 0.75% (w/w), at least 1.0% (w/w), at least, 1.5% (w/w), or at least 2.0% (w/w)of total BIAs in the reaction mixture. In some other cases, the reaction mixture comprises salutaridine, in a weight percentage of at least 0.1% (w/w), at least 0.25% (w/w), at least 0.5% (w/w), at least 0.75% (w/w), at least 1.0% (w/w), at least, 1.5% (w/w), or at least 2.0% (w/w)of total BIAs in the reaction mixture. [000180] Upon completion of the methods or reactions described throughout, the amount of a particular BIA, e.g. , thebaine or morphine, present in the reaction mixture can be less than 10.0% (w/w), less than 5.0% (w/w), less than 2.5% (w/w), less than 2.0% (w/w), less than 1.5% (w/w), or less than 1.0% (w/w) of the total BIAs in the reaction mixture. In some instances, the reaction mixture comprises thebaine in a weight percentage of less than 10.0% (w/w), less than 5.0% (w/w), less than 2.5% (w/w), less than 2.0% (w/w), less than 1.5% (w/w), or less than 1.0% (w/w) of total BIAs in the reaction mixture. In some other cases, the reaction mixture comprises salutaridinol 7-O-acetate, in a weight percentage of less than 10.0% (w/w), less than 5.0% (w/w), less than 2.5% (w/w), less than 2.0% (w/w), less than 1.5% (w/w), or less than 1.0% (w/w) of total BIAs in the reaction mixture. In some other cases, the reaction mixture comprises salutaridinol, in a weight percentage of less than 10.0% (w/w), less than 5.0% (w/w), less than 2.5% (w/w), less than 2.0% (w/w), less than 1.5% (w/w), or less than 1.0% (w/w) of total BIAs in the reaction mixture. In some other cases, the reaction mixture comprises salutaridine, in a weight percentage of less than 10.0% (w/w), less than 5.0% (w/w), less than 2.5% (w/w), less than 2.0% (w/w), less than 1.5% (w/w), or less than 1.0% (w/w) of total BIAs in the reaction mixture.

[000181] When salutaridine is made, its presence within the medium can be at a concentration of at least 75 μg/L. In some cases, salutaridine can be present within the medium at a

concentration of at least 100 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 125 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 150 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 175 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 200 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 250 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 300 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 350 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 400 μ^. In some cases, salutaridine can be present within the medium at a concentration of at least 450 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 500 μg/L.

[000182] When salutaridinol is made, its presence within the medium can be at a concentration of at least 5 μg/L. In some cases, salutaridinol can be present within the medium at a concentration of at least 10 g/L. In some cases, salutaridinol can be present within the medium at a concentration of at least 15 μg/L. In some cases, salutaridinol can be present within the medium at a concentration of at least 20 μ§/ In some cases, salutaridinol can be present within the medium at a concentration of at least 25 μg/L. In some cases, salutaridinol can be present within the medium at a concentration of at least 40 μg/L. In some cases, salutaridinol can be present within the medium at a concentration of at least 50 μg/L. In some cases, salutaridinol can be present within the medium at a concentration of at least 65 μg/L. In some cases, salutaridinol can be present within the medium at a concentration of at least 75 μg/L. In some cases, salutaridinol can be present within the medium at a concentration of at least 90 μg/L. In some cases, salutaridine can be present within the medium at a concentration of at least 100 μg/L. In some cases, salutaridinol can be present within the medium at a concentration of at least 150 μg/L. In some cases, salutaridinol can be present within the medium at a concentration of at least 200 μg L. In some cases, salutaridinol can be present within the medium at a concentration of at least 250 μg/L.

183] When thebame is made, its presence within the medium can be at a concentration of at least 250 pmol μg protein ^"1 . In some cases, thebaine can be present withm the medium at a concentration of at least 350 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 500 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 650 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 750 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 900 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 1000 pmol g protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 1250 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 1500 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 1700 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 1900 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 2000 pmol μg protein ^"1 . In some cases, thebaine can be present within the medium at a concentration of at least 2250 pmol μg protein ^"1 . In some cases, thebaine can be present withm the medium at a concentration of at least 2500 pmol μg protein ^"1 . In some cases, thebaine can be present withm the medium at a concentration of at least 5 μg/L. In some cases, thebaine can be present within the medium at a concentration of at least 10 μg/L. In some cases, thebaine can be present within the medium at a concentration of at least 12.5 μg/L. In some cases, thebaine can be present within the medium at a concentration of at least 15 μ^. In some cases, thebaine can be present within the medium at a concentration of at least 17.5 μg/L. In some cases, thebaine can be present within the medium at a concentration of at least 20 μg/L. In some cases, thebaine can be present within the medium at a concentration of at least 22.5 μg/L. In some cases, thebaine can be present within the medium at a concentration of at least 25 μι S/L. In some cases, thebaine can be present within the medium at a concentration of at least 30 μι S L. In some cases, thebaine can be present within the medium at a concentration of at least 35 μ^. In some cases, thebaine can be present within the medium at a concentration of at least 40 μι S L. In some cases, thebaine can be present within the medium at a concentration of at least 45 μι S L. In some cases, thebaine can be present within the medium at a concentration of at least 50 μι S L. In some cases, thebaine can be present within the medium at a concentration of at least 75 μι S L. In some cases, thebaine can be present within the medium at a concentration of at least 100 μg/L.

Exemplary uses of the BIAs

[000184] Preparations of BIAs {e.g., thebaine) obtained may be used for any and all uses. The BIAs can be isolated and sold as purified products. Or these purified products can be a feedstock to make additional BIAs or morphinan alkaloids.

[000185] Derivative morphinan alkaloid compounds may be used to manufacture medicinal

compounds. Thus, for example when considering thebaine, it be converted to a derivative morphinan alkaloid compound selected from oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxycodeinone, neopinone, buprenorphine, or any combination thereof

[000186] Accordingly, in one aspect, disclosed is a use of thebaine (or other BIA) as a feedstock compound in the manufacture of a medicinal compound.

[000187] The medicinal compound can be a natural derivative morphinan alkaloid compound or, in some cases, a semi-synthetic derivative morphinan alkaloid compound. For example, thebaine may be converted to oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone naltrexone, naloxone, hydroxycodeinone, neopinone, buprenorphine, or any combination thereof which each may subsequently be used to prepare a pharmaceutical formulation.

Pharmaceutical Compositions and Routes of Administration [000188] The BIAs (e.g. , thebame, morphine, and their derivatives) also include pharmaceutically acceptable derivatives or prodrugs thereof. A "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative thereof.

[000189] Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases include alkali metal (e.g. , sodium), alkaline earth metal (e.g. , magnesium), ammonium and N-(alkyl) ₄ ⁺ salts.

[000190] For preparing pharmaceutical compositions from the compounds of the present

invention, pharmaceutically acceptable carriers include either solid or liquid carriers. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA.

[000191] In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[000192] Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxy propylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilizmg agents are added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

[000193] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution. [000194] The pharmaceutical preparation can be a unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[000195] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

[000196] The disclosure is now described with reference to the following Examples. These

Examples are provided for the purpose of illustration only and the disclosure should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

EXAMPLES

Example 1 - Preparation of Genetically Modified Microorganisms that is capable of making high levels of salutaridine, salutaridinol and thebaine

[000197] The S. cerevisiae strain CEN.PK were transformed with nucleic acids encoding for a variety of enzymes that are capable of or take part in the fermentation of sugar to BIAs, such as thebaine. The genotypes of the strains are found in FIG. 4A. The strains contained up to nine chromosomally integrated plant genes encoding BIA biosynthetic enzymes capable of converting (<S)-norlaudanosoline to salutaridine, salutaridinol 7-<9-acetate, or thebaine. Strain Sc- 2 harbored the first seven biosynthetic genes, resulting in the production of salutaridine. Strain Sc-3 contained two additional genes, encoding SalR and SalAT, leading to the formation of salutaridinol 7-O-acetate, whereas strain Sc-4 also included SalR, SalAT and THS2 gene. The sequences of these constructs are presented as SEQ ID NOs. 58 to 63.

Example 2 - Hydroxylated Byproducts of Salutaridinol 7-O-acetate

[000198] A longstanding dilemma in thebaine biosynthesis involved the formation of the

hydrofuran ring in the precursor intermediate salutaridinol. Although salutaridinol undergoes spontaneous allylic elimination at pH <5 in vitro to yield thebaine (FIG. 2A), the possibility that the cyclization of salutaridinol was an enzymatic process remained to be determined. All indications suggested that the cyclization of salutaridinol into thebaine was a spontaneous reaction. First, it was suggested that the C7 hydroxyl of salutaridinol must be functionalized to furnish a better leaving group for intramolecular SN2' syn displacement. This prediction was realized with the later discovery of salutaridinol 7-O-acetyltransferase (SalAT), which catalyzes the formation of the unstable intermediate salutaridinol 7-O-acetate At pH 8-9 in vitro, but apparently not at lower pH, thebaine was reported to form spontaneously through allylic rearrangement of salutaridinol 7-O-acetate. Second, the initially regarded precursor to thebaine (7R)-salutaridinol possessed a 'problematic' 7R-configuration, which confounded theories of a one-step, SN2' -based mechanism that could only be overcome via an enzyme-bound intermediate. The stereochemistry of biogenic salutaridinol was later corrected to the 7S- conformer, which dispensed with the theoretical requirement for a thebaine synthase enzyme. These findings drove the longstanding and dogmatic hypothesis that thebaine was formed via (i) acetylation of (7S)-salutaridinol by SalAT, and (ii) spontaneous rearrangement to thebaine occurring in a specialized cellular compartment furnished with a basic pH of 8-9 (FIG. 2A). 199] Despite all indications that this conversion was spontaneous, we continued to search for an additional (or alternative) enzyme capable of thebaine formation using either (7S)- salutaridinol 7-O-acetate or (7S)-salutaridinol. We focused our studies on regions of the opium poppy that did not exhibit elevated pH, for example, the cytoplasm of laticifers, the specialized opium poppy cells containing alkaloid-rich 'latex', displayed a neutral pH. Incubation of salutaridinol 7-O-acetate at pH 7 yielded little or no thebaine. Our own experiments involving recombinant, purified SalAT showed <10% conversion of (7S)-salutaridinol and acetyl-CoA to thebaine at pH 7. Interestingly, previous work suggested the formation of an azonine byproduct from salutaridinol 7-O-acetate between pH 6-7. However, we could not detect such a compound, but instead detected the formation of a byproduct with an exact mass corresponding to the elemental composition of (7S)-salutaridinol (C ₁₉ H ₂₃ NO ₄ ) yet exhibiting a unique retention time by reverse phase HPLC (FIG. 2A; FIG. 2B; FIG. 2D). This highly labile alkaloid byproduct with ionic m/z 330 formed spontaneously from (7S)-salutaridinol 7-O-acetate in aqueous conditions. Upon SalAT-catalyzed formation of salutaridinol 7-O-acetate, SN2' allylic elimination in the presence of water led to the formation of a hydroxylated byproduct with m/z 330 (FIG. 2C and 2E). We also recognized that a SN2 mechanism would yield the alkaloid (7R)-salutaridinol, which is unstable and difficult to purify. High resolution MS" analysis of the byproduct revealed important similarities with both (7S)-salutaridinol and C14-hydroxylated opiates such as oxycodone, indicating that m/z 330 was a hydroxylated elimination product of (7S)-salutaridinol 7-O-acetete (FIG. 2B; FIG. 2D; FIG. 2E).

[000200] High-resolution mass spectral fragmentation analysis of alkaloid byproduct (m/z 330) was performed. High-resolution MS ² collision-induced dissociation (CID) spectrum of the m/z 330-byproduct is presented in FIG. 2B. Fragmentation at 35% normalized collision energy (NCE) was conducted in the linear ion trap portion of an LTQ-Orbitrap XL instrument (Thermo Scientific) followed by full-scan FTMS detection in the Orbitrap MS (m/z 90-340). The spectrum represents an average of 161 individual scans captured over 5 minutes of continuous sample infusion (5 μί/ητίη). Ionization was performed by ESI at room temperature. Mass error was < 2 ppm across all MS" datasets, allowing reliable prediction of elemental formula (FIG. 2D). The expanded region (m/z 300-340) highlights the occurrence of a fragment with m/z 312, possibly a dehydration product of the parent ion.

[000201] MS" analysis reveals similarities and differences between (7<S)-salutaridinol and the m/z 330-byproduct. FIG. 2C shows MS" on the m/z 330-byproduct yielded ions (shown on the right). Although some byproduct ions were common with those obtained from similar (75)- salutaridinol analysis (a, f, g), others (c, d, e, h) were not. Conversely, ions (shown on the left) were unique to (75)-salutaridinol MS ⁿ (/ ^' , j, k, I, m, n) and were not observed in the m/z 330- byproduct spectra. An important fragmentation route for (7<S)-salutaridinol is amine loss through cleavage of the piperidine ring (-CH ₂ CHNHCH ₃ ), which was not evidenced in the case of m/z 330-byproduct. Instead the byproduct decomposed to fragment a by loss of a methylamine (- CH ₃ NH ₂ ), leading to the formation of other observed ions. The presence of fragment b in m z 330-byproduct MS ² spectra could result from a loss of water, a fragmentation route common to 14C-hydroxylated opiates. Fragment b could not be isolated in MS ³ spectra.

Example 3 - Thebaine Synthase

[000202] Kinetic analysis of THS2 was performed using a direct assay with (7S)-salutaridinol 7- O-acetate as the substrate. (7<S)-Salutaridinol 7-O-acetate is relatively stable in chloroform but exposure to aqueous conditions caused the rapid (< 2 min) and substantial (>70%) formation of m/z 330-byproduct. To minimize hydroxylated byproduct formation and maintain linear range parameters, the assay was quenched with chloroform after 30 sec (FIG. 3A). The small amount of thebaine formed spontaneously in the absence of THS2 was subtracted from assays containing active enzyme. THS2 displayed positive cooperativity (η = 2.3) with a A of 20 μΜ, a Vmax of 4.0 nmol min ^"1 μg ^"1 , and a pH optimum of 8.0 (FIG. 3B, FIG. 3C, and FIG. 5). These results suggested that the THS homodimer exhibits allostery with respect to its substrate. The enzymatic reaction mechanism for thebaine formation likely involves deprotonation of the C4 hydroxyl group of (7<S)-salutaridinol 7-O-acetate by a catalytic residue acting as a general base, yielding a phenolate anion nucleophile. This newly formed nucleophile would then attack at C5 to facilitate S _N 2' syn displacement of the 0-acetyl leaving group (FIG. 2E). This mechanism has similarities with those observed for S-adenosylmethionine (SAM)-dependent O- methyltransferases. For example, norcoclaurine 6-6>-methyltransferase (60MT) in BIA biosynthesis uses a catalytic histidine to abstract a hydroxyl proton prior to S _N 2 attack of SAM. However, important differences of the THS reaction compared with ( -methylation are the intramolecular, rather than intermolecular, C-0 coupling and the lack of a cofactor. THS2 did not produce thebaine directly from (7S)-salutaridinol at pH 7.0, although activity on (75)- salutaridinol 7-O-acetate was marginally inhibited by the addition of salutaridine and (75)- salutaridinol. In comparison, two acetylated BIA derivatives showed little inhibitory effect. The concomitant production of thebaine and suppression of hydroxylated byproduct formation by THS could reflect 'shielding' of the labile O-acetate from intermolecular nucleophilic attack by OH ^" ions in aqueous solution. Structural elucidation will reveal if a base-acting, catalytic residue is required for activity, or whether THS simply acts as a 'shield' against OH ^" attack, which precludes the competing reaction and facilitates spontaneous thebaine formation. Similar intramolecular S _N 2' displacement is thought to occur elsewhere in natural product biosynthesis, such as in the formation of the meroterpenoid rossinone B by sea squirts. It is not known whether closure of the tetrahydrofuran ring in rossinone B occurs spontaneously, or requires an enzyme. The requirement of not just one, but two enzymes for the biological formation of thebaine from (7<S)-salutaridinol, which occurs spontaneously in vitro at pH <5, attests to the difficulty of the reaction.

[000203] Three strains of S. cerevisiae were used to evaluate the impact of THS on reticuline, salutaridine, salutaridinol, and thebaine yields in engineered yeast (FIG. 4A and FIG. 4B). The strains contained up to nine chromosomally integrated plant genes encoding BIA biosynthetic enzymes capable of converting (S)-norlaudanosoline to salutaridine, salutaridinol 7-O-acetate, or thebaine (FIG. 4A). Strain Sc-2 harbored the first seven biosynthetic genes, resulting in the production of salutaridine. Strain Sc-3 contained two additional genes, encoding SalR and SalAT, leading to the formation of salutaridinol 7-O-acetate, whereas strain Sc-4 also included SalR, SalAT and THS2 genes (see SEQ ID NOs. 58 to 63).

[000204] One of two plasmids was also transformed into each strain. The pEV-1 vector served as the empty-vector negative control (SEQ ID NO. 64), whereas pTHS2 provided enhanced expression of THS2 in addition to the chromosomally integrated copy in Sc-4 (SEQ ID NO. 65). Both integrated and plasmid-expressed genes were under the control of galactose-inducible promoters to ensure coordinated expression. Yeast was cultured using standard protocols with the addition of 2.5 mM CR/S)-norlaudanosoline to the induction medium, followed by a 96-hour fermentation at 30°C. Strain Sc-2, produced reticuline and salutaridine, but did not yield salutaridinol or thebaine with or without the addition of pTHS2 (FIG. 4B). In contrast, strain Sc- 3 transformed with pTRV2 showed decreased levels of salutaridine, and produced 479 ± 62 μg/L thebaine after 96 hours, compared with only 29 + 3 μg/L in the control. Thebaine levels in strain Sc-4 harboring chromosomally integrated THS2 were further elevated after 96 hours to 439 + 36 μg/L in the control and 699 + 44 μg/L with the addition of pTRV2. Levels of the hydroxylated byproduct showed an inversely proportional decrease compared with the increased accumulation of thebaine. The lack of a directly inverse relationship between thebaine and the hydroxylated byproduct can be attributed to the instability of the latter compound(s).

Example 4 - The effect of pH on BIA production

[000205] The strains from FIG. 4A were grown up in Standard Synthetic Media (SE media)

(containing 2% glucose) until they reached a sufficient OD. After reaching proper OD, the strains were transferred into modified 96 well plates with medium containing 2% galactose, 1.8% raffinose, 0.2% glucose. The strains were also given a ImM R-reticuline feed. The pH within the wells were also adjusted from 3.5 to 9.2 from left to right. FIG. 7B shows the experimental design.

[000206] As shown in FIG. 8, higher pH showed greater increases in salutaridine and salutaridinol levels. The highest levels of salutaridine were seen at a pH of 7.5 and above. The highest levels of salutaridinol were observed at a pH of between 5.5. and 6.5. Thebaine levels were seen to peak above a pH of 6.0.

Example 5 - The effect of pH on salutaridinol and thebaine production

[000207] The strain from example 4 were grown up in SE media supplemented with G418 until they reached a sufficient OD. After reaching proper OD, the strains were transferred into modified 96 well plates with medium containing 2% galactose, 2% raffmose, 0.2% glucose. The strains were also given a ImM R-reticuline or ImM salutaridine feed for 24 or 48 hours. The pH within the wells were also adjusted to 6.5 and 7.5 along with a negative control (no pH adjustment).

[000208] As shown in FIG. 9A, when the strains from example 3 were fed a reticuline feed,

strains having a media pH adjustment to 7.5 exhibited the largest gain of both salutaridinol and thebaine titers compared to the negative control. Strains grown in media having a pH of 6.5 did show vast improvement in salutaridinol and thebaine titers compared to negative controls. Further, both the strains that were grown in pH 6.5 and 7.5 showed a significant increase of salutaridinol and thebaine titers after 48 hours, compared to respective production titers after 24 hours. There was no increase in salutaridinol or thebaine titers between 24 and 48 hours for the negative control.

[000209] As shown in FIG. 9B, when the strains from example 3 were fed a salutaridine feed, strains having a media pH adjustment to 7.5 exhibited a very large gain of salutaridinol and a more modest gain thebaine titers compared to the negative control. Strains grown in media having a pH of 6.5 did show a significant improvement in salutaridinol and thebaine titers compared to negative controls. Further, both the strains that were grown in pH 6.5 and 7.5 showed a significant increase of salutaridinol and thebaine titers after 48 hours, compared to respective production titers after 24 hours. There was no increase in salutaridinol or thebaine titers between 24 and 48 hours for the negative control.

Example 6 - The effect of media on thebaine production

[000210] The strain from example 4 were grown up in either SE media or YPD media ("yeast extract peptone dextrose" which contains yeast extract, peptone, double-distilled water, and glucose or dextrose) both supplemented with G418 until they reached a sufficient OD. After reaching proper OD, the strains were transferred into modified 96 well plates with their respective medium containing 2% galactose, 2% raffmose, 0.2% glucose. The strains were also given a ImM R-reticuline or ImM salutaridine feed for 24 or 48 hours. The pH within the wells were also adjusted to 6.5 and 7.5 along with a negative control (no pH adjustment).

[000211] As shown in FIG. 10, when starting from a reticuline feed, the strain grown in pH 7.5 exhibited increased thebaine titers (normalized to OD) when compared to negative controls. The strain grown in pH 6.5 also showed increased thebaine titers when compared to negative controls. However, strains grown in YPD media exhibited higher thebaine titers when compared to strains grow in SE media. These increased titers between different media were seen in all groups, including the negative control, pH 6.5 and pH 7.5.

[000212] As shown in FIG. 10, when starting from a salutaridine feed, the strain grown in pH 7.5 exhibited a significant increase in thebaine titers (normalized to OD) when compared to negative controls. The strain grown in pH 6.5 also showed increased thebaine titers when compared to negative controls. However, strains grown in SE media exhibited much higher thebaine titers (about 100% or more) when compared to strains grow in YPD media. These increased titers between different media were seen in all groups, including the negative control, pH 6.5 and pH 7.5

Example 7 - pH control starting from L-DOPA to thebaine

[000213] The strain from example 4 (FIG. 4A) were grown up in SE media supplemented with G418, 2% galactose, 1.8% raffinose, and 0.2% glucose. The strains were fed with 5mM L- DOPA, lOmM methionine, and lOmM sodium ascorbate. The strains were i) harvested for analysis at 24 hours (having a pH of < 6.0); ii) supplemented with a 1 : 10 volume of water (negative control) or 1M MES (up to pH 7.5). After 24 additional hours (48 hours today), the cultures were harvested and analyzed.

[000214] As shown in FIG. 11, the strains harvested at 24 hours (having a pH of < 6.0) produced similar reticuline titers between the various strains (strains 1, 2, and 3). Strains harvested at 48 hours that were buffered with the negative control exhibited similar reticuline titers. However, all strains that were cultured in pH 7.5, produced higher reticuline titers, of approximately 25% to 30%.

[000215] As shown in FIG. 12, titers of salutaridine, salutaridmol, and these were measured in these strains. Strain 1 (strains having enzymes from DODC to SalSyn) produced very high levels of salutaridine both after 24 hours and 48 hours supplemented with water (negative control). However, after 48 hours at pH 7.5, strain 1 produced high levels of salutaridine, approximately 25% more than its negative control or after 24 hours. Strain 1 failed to produce any detectable levels of salutaridinol or thebaine, since the enzymes that perform those reactions were not present in strain 1.

[000216] Strain 2 (strains having enzymes from DODC to SalSyn, plus pGAL SalR and pTEAl SalAT) showed elevated levels of salutaridine when cultured in pH 7.5 for 48 hours. Strains cultured for 24 hours (having a pH of < 6.0) and cultured for 48 hours diluted in water (negative control) demonstrated lower salutaridine levels (approximately 30% decrease). Strain 2 also produced salutaridinol and thebaine at similar levels at 24 hours, 48 hours (negative control) and 48 hours in pH 7.5.

[000217] Strain 3 (strains having enzymes from DODC to SalSyn, plus pGAL SalR and pTEAl SalAT plus BETV1-A) produced slightly elevated levels of salutaridine only when cultured at pH 7.5 after 48 hours. Salutaridinol levels were also mostly unchanged at 24 hours, 48 hours (negative control) and 48 hours in pH 7.5. However, thebaine levels increased (approximately 40%) when culturing in pH 7.5 for 48 hours, compared to 24 hour culture and negative control cultures. [000218] As shown in FIG. 13, OD levels were similar between the same strain at 24 hours, 48 hours (no buffer), and 48 hours (with buffering - pH 7.5).

[000219] In summary, without buffering, there was no significant observed difference between titers of BIA pathway compounds at 24 or 48 hours. However, when buffered to pH 7.5, reticuline titers increased approximately 1.5-fold between 24 and 48 hours. Thebaine titers also increased approximately 1.4-fold between 24 and 48 hours. No significant same strain OD differences were observed between buffered and not buffered cultures.

Example 8 - pH control at 24 and 48 hours

[000220] To determine the length of pH adjustments, strain: Y16_T7 (which has the following integrated genes Pbra60MT, CjapCNMT, Psom40MT, REPI-2, PbraSalSyn, PbraCPRl, PsomSalR, PsomSalAT, and PsomBetvl-1) were grown in three conditions. In the first condition, the media was left unbuffered. In the second condition, buffer was added to the media at 24 h to adjust the pH to 7.5. In the third condition, buffer was added to the media at 48 h to adjust the pH to 7.5. The optimum condition to produce thebaine was when the pH was adjusted after 24h compared to the other two conditions, suggesting pH control and the timing of pH control are important for thebaine production.

Example 9 - pH controls BIAs production in the presence of Betvl

[000221] Salutaridine, thebaine, and hydroxylated by-product (mz330) titers were measured using the yeast strain Y16_T9 (which has the following integrated genes Pbra60MT, CjapCNMT, Psom40MT, REPI-2, PbraSalSyn, PbraCPRl, PsomSalR, PsomSalAT, and PsomBetvl-1). Controls were transformed with either Empty Vector (EV) and test strains were transformed with a high copy plasmid containing HA-Betvl (N-terminal HA epitope tag). Levels of salutaridine, thebaine, and the hydroxylated by-product (mz330) were measured. As show in FIG. 15A, salutaridine titers increased by 4-fold at pH 7.5 compared to the unbuffered control or at pH 6.5. Salutaridine titers were unaffected by the presence or absence of Betvl.

[000222] by-product (mz330) titers were measured using the yeast strain Y16_T9 (which has the following integrated genes Pbra60MT+ CjapCNMT+Psom40MT + REPI-2 + PbraSalSyn + PbraCPRl + PsomSalR + PsomSalAT). Controls were transformed with either Empty Vector (EV) and test strains were transformed with a high copy plasmid containing HA-Betvl (N- terminal HA epitope tag). Levels of salutaridine, thebaine, and the hydroxylated by-product (mz330) were measured. As show in FIG. 15A, salutaridine titers increased by 4-fold at pH 7.5 compared to the unbuffered control or at pH 6.5. Salutaridine titers were unaffected by the presence or absence of Betvl. [000223] On the other hand, FIG. 15B shows that thebaine titers at pH 6.5, pH 7.5, and unbuffered control in the presence of Betvl. Thebaine production in the presence of Betvl, increased approximately 5-fold at pH 7.5 compared to pH 6.5 and unbuffered control. In other words, at pH 7.5, thebaine titers were significantly higher than at pH 6.5.

[000224] FIG. 15C shows m/z 330 levels at pH 6.5, pH 7.5, and unbuffered control in the

presence and absence of Betvl . m/z 330 levels dropped approximately 50% in the presence of Betvl at pH 6.5, pH 7.5, and in unbuffered conditions.

Example 10 - pH control of Cytochrome P450 Reductase (CPR) during the formation of BIAs

[000225] Two strains with slightly differing genotypes were used in the fermentation of

salutaridine and reticuline. These two strains were fermented in differing pH conditions. Each strain contained three methyltransferases (60MT, CNMT, and 40MT), one NMCH, two variants of SalSyn, and a reticuline epimerase (REPI). In addition, the APY254 strain expressed a P. somniferum CPR and a second copy of NMCH, whereas the APY299 expressed an A. annua CPR. When fed with either NCC or NLDS without buffer, both strains produced similar levels of combined reticuline and salutaridine and less than half was salutaridine (FIG.

16A). However, when the media was buffered to pH 7.5 with HEPES, total production from an

NCC feed increased up to 14x and the majority of the product was salutaridine (FIG.

16B). Production from an NLDS feed also increased up to 3x with a similar increase ratio of salutaridine to reticuline (FIG. 16B). These results indicate the media conditions promote increased total reticuline and salutaridine production while also increasing the relative salutaridine to reticuline ratio. These results also suggest that CPR's enzymatic reactions are more efficient at a higher pH.

Example 11 - pH controls BIAs production

[000226] One strain (yGPVR 151) was used in the fermentation of reticuline in differing pH

conditions. Levels of dopamine (FIG. 17B), and reticuline (FIG. 17C) were measured. Realtime pH values were also collected during the fermentation (FIG. 17A). pH was regulated by one-sided by addition of NH ₄ OH base to keep the pH level over pH 4.0. Highest reticuline production and dopamine consumption were observed in case of the highest pH profile (over pH 5.0).

Example 12 - pH and dissolved oxygen control BIAs production

[000227] One strain (yGPVR 251) was used in the fermentation of salutaridine and reticuline in differing pH and dissolved oxygen (PO ₂ ) conditions. Levels of dopamine (FIG. 18B), reticuline (FIG. 18C) and salutaridine (FIG. 18D) were measured. Real time pH values were also collected during the fermentation (FIG. 18A). In one set, one-sided pH regulation was applied by addition of NH ₄ OH base to keep pH level over pH 6.0 from the start of fermentation, while for other runs only initial pH was set to 5.5 and NH ₄ OH base addition was applied to keep pH level over pH 4.0 during initial phase (until 24 hours). Highest salutaridine production and lowest dopamine and reticuline accumulation was observed in case of pH control to exceed pH 6.0 value. Lower (7%) dissolved oxygen level caused slightly lower salutaridine titer than 20%.

[000228] Example 13 - Different pH setpoints affect BIAs production

[000229] Two strains (yGPVR 352 and 353) were used in the fermentation of salutaridine and reticuline in differing pH conditions. Levels of dopamine (FIG. 19B), reticuline (FIG. 19C) and salutaridine (FIG. 19D) were measured. Real time pH values were also collected during the fermentation (FIG. 19A). In two parallel sets, one-sided pH regulation was applied by addition of NH ₄ OH base to keep pH level over pH 6.0 and pH 6.5 values with yGPVR 353 strain;

yGPVR 352 strain was tested in a fermentation with pH control to keep pH value over pH 6.0 in two parallel runs. Highest salutaridine production and lowest dopamine and reticuline accumulation was observed in case of pH control to pH 6.0 value for both strain. If pH exceeded pH 8.0 value, bioconversion stopped.

[000230] Example 14 - Different pH setpoints and two-sided pH regulation affect BIAs production

[000231] One strain (yGPVR 454) was used in the fermentation of salutaridine and reticuline in differing pH conditions. Levels of dopamine (FIG. 20B), reticuline (FIG. 20C) and salutaridine (FIG. 20D) were measured. Real time pH values were also collected during the fermentation (FIG. 20A). In two parallel sets pH regulation was applied in one-sided form by addition of NH ₄ OH base to keep pH level over pH 6.0, while in two parallel sets pH regulation was applied in two-sided form by addition of NH ₄ OH and HC1 solutions to maintain pH level at pH 6.0. Higher dopamine and reticuline accumulation and lower salutaridine production was observed in case of pH control to pH 6.0 by two-sided pH regulation, while keeping pH above pH 6.0 value caused better bioconversion from dopamine to reticuline and from reticuline to salutaridine. Example 15 - Different pH setpoints and pH shift during fermentation affect BIAs production

[000232] One strain (yGPVR 454) was used in the fermentation of salutaridine and reticuline in differing pH conditions. Levels of dopamine (FIG. 21B), reticuline (FIG. 21C), reticuline S (FIG. 21D), reticuline R (FIG. 21E), and salutaridine (FIG. 21F) were measured. Real time pH values were also collected during the fermentation (FIG. 21A). In two parallel sets pH regulation was applied in two-sided form by addition of NH ₄ OH and HCl solutions to keep pH level at pH 6.0; in two parallel sets pH regulation was applied in two-sided form by addition of NH ₄ OH and HCl solution to make pH profile in the following way: pH 6.0 from start of fermentation until 16-18 hours, ramping up pH to 7.5 until 20 hours, ramping down pH to 6.5 until 60 hours and keep pH at 6.5 until end of fermentation; in two parallel sets pH regulation was applied in two-sided form by addition of NH ₄ OH and HCl solutions to keep pH at pH 6.0 value until 72 hours and shifted up to pH 6.5; in two parallel sets pH regulation was applied in two-sided form by addition of NH ₄ OH and HCl solutions to keep pH at pH 6.0 value and dissolved oxygen level (p(¾) was kept at 20% until 72 hours and shifted down to 7%. Highest effect on salutaridine titer and conversion of dopamine and reticuline was observed in case of pH shift from 6.0 to 6.5 at 72 hours.