Methods of producing morphinan alkaloids and derivatives

1 . An engineered non-plant cell, comprising:

i) an epimerase,

ii) a thebaine synthase; and

iii) at least one modification selected from the group consisting of: a) a substrate inhibition alleviating mutation, b) a product inhibition alleviating mutation, c) a cofactor recovery promoting mechanism, d) a feedback inhibition alleviating mutation, e) transcriptional modulation modification, and f) an inactivating mutation; and

iv) wherein the cell further comprises three or more of the enzymes of:

(a) tyrosinase or tyrosine hydroxylase (TYR or TyrH), L-DOPA decarboxylase (DODC), 6-O-methyltransferase (6OMT), coclaurine-N-methyltransferase (CNMT), N-methylcoclaurine 3′-hydroxylase (CYP80B1), 4′-O-methyltransferase (4′OMT), salutaridine synthase (SalSyn), salutaridine reductase (SalR), salutaridinol 7-O-acetyltransferase (SalAT), and norcoclaurine synthase (NCS); or

(b) TYR or TyrH, DODC, monoamine oxidase (maoA), 6OMT, CNMT, 4′OMT, DRS-DRR, SalSyn, SalR, SalAT, and norcoclaurine synthase (NCS), and

wherein, within the engineered non-plant cell, the engineered non-plant cell converts a precursor of a promorphinan molecule to an alkaloid product selected from the group consisting of: i) a morphinan alkaloid, ii) a nal-opioid alkaloid, and iii) a nor-opioid alkaloid.

2 . The engineered non-plant cell of claim 1 , wherein the epimerase is an engineered epimerase.

3 . The engineered non-plant cell of claim 2 , wherein the engineered epimerase is a split epimerase.

4 . The engineered non-plant cell of claim 2 , wherein the engineered epimerase converts an (S)-l-benzylisoquinoline precursor to an (R)-l-benzylisoquinoline product.

5 . The engineered non-plant cell of claim 4 , wherein the engineered epimerase converts(S)-reticuline to (R)-reticuline.

6 . The engineered non-plant cell of claim 4 , wherein at least 50% of the (S)-l-benzylisoquinoline alkaloid molecules within the engineered non-plant cell are converted to the (R)-l-benzylisoquinoline product.

7 . The engineered non-plant cell of claim 1 , wherein the thebaine synthase is an engineered thebaine synthase.

8 . The engineered non-plant cell of claim 1 , wherein the precursor of a promorphinan molecule is fed to the engineered non-plant cell.

9 . The engineered non-plant cell of claim 1 , wherein the precursor of a promorphinan molecule is produced within the engineered non-plant cell.

10 . The engineered non-plant cell of claim 1 , wherein the precursor of a promorphinan molecule is selected from the group consisting of reticuline, 3′hydroxy-N-methylcoclaurine, coclaurine, norcoclaurine, norlaudanosoline, methylnorlaudanosoline, laudanosoline, methylnorlaudanosoline, norreticuline, 3′hydroxy-N-methylcoclaurine, 4′-0′-methylaudanosoline, L-Dopa, tyrosine, dopamine, 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA), hydroxyphenylpyruvate, prephenate, chorismate, 5-enolpyruvylshikimate-3-phosphate (EPSP), 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), erythrose-4-phosphate (E4P), phosphoenolpyruvate (PEP), and glucose.

11 . The engineered non-plant cell of claim 1 , wherein at least 50% of tetracyclic promorphinan precursor molecules within the engineered non-plant cell are converted to thebaine.

12 . The engineered non-plant cell of claim 1 , wherein the precursor of a promorphinan molecule is an (S)-substrate of Formula I:

or a salt thereof, wherein:

R 1 , R 2 , R 3 , and R 4 are independently selected from hydrogen and methyl; and

R 5 is selected from hydrogen, hydroxy, and methoxy.

13 . The engineered non-plant cell of claim 12 , wherein at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is hydrogen.

14 . The engineered non-plant cell of claim 1 , wherein the precursor of a promorphinan molecule is (S)-substrate is a compound of Formula II:

or a salt thereof, wherein:

R 3 is selected from hydrogen and C 1 -C 4 alkyl;

R 6 and R 7 are independently selected at each occurrence from hydroxy, fluoro, chloro, bromo, carboxaldehyde, C 1 -C 4 acyl, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy;

n is 0, 1, 2, 3, or 4; and

n′ is 0, 1, 2, 3, 4 or 5.

15 . The engineered non-plant cell of claim 1 , wherein the precursor of a promorphinan molecule is selected from tyrosine and sugar.

16 . The engineered non-plant cell of claim 1 , further comprising at least one modification selected from the group consisting of: i) a BIA-generating modification, ii) an O-demethylation modification, iii) an N-demethylation modification, and iv) an N-linked modification.

17 . The engineered non-plant cell of claim 1 , wherein the morphinan alkaloid product is a thebaine, codeinone, codeine, morphine, morphinone, oripavine, neopinone, neopine, neomorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone, oxycodone, 14-hydroxycodeine, morphinone, hydromorphone, dihydromorphine, dihydroetorphine, ethylmorphine, etorphine, metopon, buprenorphine, pholcodine, heterocodeine, or oxymorphone.

18 . The engineered non-plant cell of claim 1 , wherein (i) the nal-opioid alkaloid product is a naltrexone, naloxone, nalmefene, nalorphine, nalorphine, nalodeine, naldemedine, naloxegol, 6b-naltrexol, naltrindole, methylnaltrexone, methylsamidorphan, alvimopan, axelopran, bevenpran, dinicotinate, levallorphan, samidorphan, buprenorphine, dezocine, eptazocine, butorphanol, levorphanol, nalbuphine, pentazocine, phenazocine, norbinaltorphimine, or diprenorphine or (ii) the nor-opioid alkaloid product is a norcodeine, noroxycodone, northebaine, norhydrocodone, nordihydro-codeine, nor-14-hydroxy-codeine, norcodeinone, nor-14-hydroxy-codeinone, normorphine, noroxymorphone, nororipavine, norhydro-morphone, nordihydro-morphine, nor-14-hydroxy-morphine, normorphinone, or nor-14-hydroxy-morphinone.

19 . The engineered non-plant cell of claim 1 , wherein the engineered non-plant cell is a bacterial cell or a fungal cell.

20 . A method of converting a precursor of a promorphinan molecule to a thebaine, or a derivative thereof, comprising:

contacting the precursor of a promorphinan molecule with an epimerase and a thebaine synthase, wherein at least one precursor of promorphinan molecule is produced within an engineered non-plant cell where the conversion occurs,

wherein the cell further comprises three or more of the enzymes of:

(a) tyrosinase or tyrosine hydroxylase (TYR or TyrH), L-DOPA decarboxylase (DODC), 6-O-methyltransferase (6OMT), coclaurine-N-methyltransferase (CNMT), N-methylcoclaurine 3′-hydroxylase (CYP80B1), (4′-O-methyltransferase) (4′OMT), salutaridine synthase (SalSyn), salutaridine reductase (SalR), salutaridinol 7-O-acetyltransferase (SalAT), and norcoclaurine synthase (NCS); or

(b) TYR or TyrH, DODC, monoamine oxidase (maoA), 6OMT, CNMT, 4′OMT, DRS-DRR, SalSyn, SalR, SalAT, and norcoclaurine synthase (NCS) and

wherein contacting the precursor of a promorphinan molecule with the epimerase and thebaine synthase converts the precursor of a promorphinan molecule to a thebaine, or a derivative thereof, within the engineered non-plant cell.