Methods of producing morphinan alkaloids and derivatives

An engineered microbial cell, comprising:

• i) an engineered epimerase, wherein the engineered epimerase converts an (S)-1-benzylisoquinoline precursor to an (R)-1-benzylisoquinoline product within the engineered microbial cell;
• ii) a thebaine synthase, wherein the thebaine synthase has measurable activity within the engineered microbial cell;
• 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 the enzymes of:
• (a) tyrosinase or tyrosine hydroxylase (TYR or TyrH), L-DOPA decarboxylase (DODC), 6-O-methyltransferase (6OMT), coclaurine-N-methyltransferase (CNMT), cytochrome P450 80B1 (CYP80B1), (4′-O-methyltransferase) (4′OMT), dehydroreticuline synthase and dehydroreticuline reductase (DRS-DRR), salutaridine synthase (SalSyn), salutaridine reductase SalR), and salutaridinol 7-O-acetyltransferase (SalAT);
• or
• (b) TYR or TyrH, DODC, monoamine oxidase (maoA), 6OMT, CNMT, 4′OMT, DRS-DRR, SalSyn, SalR, and SalAT, and
• wherein, within the engineered microbial cell, the engineered microbial cell converts a plurality of tetracyclic promorphinan precursor molecules to a category of alkaloid products selected from the group consisting of:
• i) a morphinan alkaloid, ii) a nal-opioid alkaloid, and iii) a nor-opioid alkaloid, and
• wherein the engineered microbial cell produces at least 10% more thebaine relative to a same microbial cell that lacks the thebaine synthase.