Methods of producing morphinan alkaloids and derivatives
Inventors
Smolke, Christina D. • Trenchard, Isis • Hawkins, Kristy M. • Thodey, Catherine
Assignees
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Publication Number
US-11884949-B2
Publication Date
2024-01-30
Expiration Date
Abstract
A method of producing promorphinan, morphinan, nal-opioid, and nor-opioid alkaloid products through the increased conversion of a promorphinan alkaloid to a morphinan alkaloid. The method comprises contacting the promorphinan alkaloid with at least one enzyme. Contacting the promorphinan alkaloid with the at least one enzyme converts the promorphinan alkaloid to a morphinan alkaloid.
Core Innovation
The invention relates to an engineered microbial cell that converts tetracyclic promorphinan precursor molecules to alkaloid products selected from morphinan alkaloid, nal-opioid alkaloid, and nor-opioid alkaloid categories. The engineered microbial cell includes an engineered epimerase that converts an (S)-1-benzylisoquinoline precursor to an (R)-1-benzylisoquinoline product within the engineered microbial cell, and a thebaine synthase having measurable activity within the engineered microbial cell.
The engineered microbial cell further comprises at least one modification selected from substrate inhibition alleviating mutation, product inhibition alleviating mutation, a cofactor recovery promoting mechanism, feedback inhibition alleviating mutation, transcriptional modulation modification, and an inactivating mutation. The cell also comprises enzymes selected from tyrosinase or tyrosine hydroxylase, L-DOPA decarboxylase, 6-O-methyltransferase, coclaurine-N-methyltransferase, cytochrome P450 80B1, 4′-O-methyltransferase, dehydroreticuline synthase and dehydroreticuline reductase, salutaridine synthase, salutaridine reductase, and salutaridinol 7-O-acetyltransferase, with an alternative set including monoamine oxidase in place of the tyrosinase component.
The engineered microbial cell produces at least 10% more thebaine relative to a same microbial cell that lacks the thebaine synthase. The described precursor and product scope is refined by selecting particular (S)-substrates and specifying stereochemistry and substituent-defined precursor structures using Formula I or Formula II, including salts, and by producing morphinan alkaloids selected from a listed set of named morphinan compounds, including thebaine.
Claims Coverage
The independent claim includes five inventive features covering stereo conversion of benzylisoquinoline precursors via an engineered epimerase, measurable thebaine synthase activity, selected modifications for inhibition alleviation, cofactor recovery, transcriptional modulation, or inactivation, specified downstream enzymes to convert tetracyclic promorphinan precursors into morphinan, nal-opioid, or nor-opioid alkaloid categories, and an increased thebaine production benchmark of at least 10%.
Engineered epimerase converts (S)-1-benzylisoquinoline precursor to (R)-1-benzylisoquinoline product
The engineered microbial cell includes an engineered epimerase that converts an (S)-1-benzylisoquinoline precursor to an (R)-1-benzylisoquinoline product within the engineered microbial cell.
Thebaine synthase has measurable activity in the engineered microbial cell
The engineered microbial cell includes a thebaine synthase having measurable activity within the engineered microbial cell.
At least one inhibition/recovery/transcription/inactivation modification
The engineered microbial cell includes at least one modification selected from substrate inhibition alleviating mutation, product inhibition alleviating mutation, a cofactor recovery promoting mechanism, feedback inhibition alleviating mutation, transcriptional modulation modification, and an inactivating mutation.
Specified enzyme set to convert tetracyclic promorphinan precursors to morphinan/nal-opioid/nor-opioid categories
The engineered microbial cell comprises enzymes selected from tyrosinase or tyrosine hydroxylase, L-DOPA decarboxylase, 6-O-methyltransferase, coclaurine-N-methyltransferase, cytochrome P450 80B1, 4′-O-methyltransferase, dehydroreticuline synthase and dehydroreticuline reductase, salutaridine synthase, salutaridine reductase, and salutaridinol 7-O-acetyltransferase, with an alternative set including monoamine oxidase in place of the tyrosinase component, and converts a plurality of tetracyclic promorphinan precursor molecules to alkaloid products selected from morphinan alkaloid, nal-opioid alkaloid, and nor-opioid alkaloid categories.
At least 10% more thebaine than a cell lacking thebaine synthase
The engineered microbial cell produces at least 10% more thebaine relative to a same microbial cell that lacks the thebaine synthase.
The core coverage is an engineered microbial cell that combines an (S)-to-(R) benzylisoquinoline epimerase, a thebaine synthase with measurable activity, at least one selected modification class, and specified downstream enzymes enabling conversion of tetracyclic promorphinan precursors into morphinan, nal-opioid, and nor-opioid alkaloid categories, with at least a 10% increase in thebaine production versus a cell lacking the thebaine synthase.
Stated Advantages
Produces at least 10% more thebaine relative to a same microbial cell that lacks the thebaine synthase.
Documented Applications
Engineered microbial cell production/conversion of benzylisoquinoline precursors into morphinan, nal-opioid, and nor-opioid alkaloid categories, including production of thebaine.
Bioinformatic enzyme identification and platform yeast strain engineering for producing (S)-reticuline, thebaine, downstream morphinans, and semi-synthetic opioids.
Extended thebaine transformations referenced with fermentation figures.
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