Methods of producing nor-opioid and nal-opioid benzylisoquinoline alkaloids

Inventors

Smolke, Christina D. • Thodey, Catherine • Trenchard, Isis

Assignees

Antheia Inc

Abstract

A method of demethylizing an opioid to a nor-opioid is provided. The method comprises contacting an opioid with at least one enzyme. Contacting the opioid with the at least one enzyme converts the opioid to a nor-opioid. A method of converting a nor-opioid to a nal-opioid is provided. The method comprises contacting a nor-opioid with at least one enzyme. Contacting the nor-opioid with the at least one enzyme converts the nor-opioid to a nal-opioid.

Core Innovation

The invention relates to engineered non-plant cells for heterologous expression of pathway enzymes that produce nor-opioid and/or nal-opioid benzylisoquinoline alkaloid products. The method includes contacting a first opioid with at least one enzyme within the engineered non-plant cell so that the first opioid is converted to a second opioid through loss of an O-linked methyl group, followed by conversion of the second opioid to a nor-opioid through loss of an N-linked methyl group.

The engineered non-plant cell is described as carrying heterologous enzyme coding sequences for O-demethylase and/or N-demethylase and N-methyltransferase functions. The disclosure also describes engineered metabolic pathways and biocatalytic routes for generating opioid derivatives in a biosynthetic context, including conversion steps that produce nor-opioids and nal-opioids through engineered enzymatic activity.

The document additionally describes engineered pathway concepts and supporting enzymatic transformations, including CYP-COR epimerase enzymes with oxidase and reductase domains for converting (S)- to (R)-1-benzylisoquinoline alkaloids and recovery of epimerized products. It also includes examples of opioid O- and N-demethylation outcomes detected by LC-MS and LC-MS/MS, together with engineered production involving reticuline, salutaridine, and thebaine.

Claims Coverage

The document contains one independent method claim with sequential demethylation in an engineered non-plant cell. The claim set includes 2 inventive features corresponding to loss of an O-linked methyl group to form a second opioid and loss of an N-linked methyl group to form a nor-opioid, with dependent refinements including host type, pathway starting point, positional limitation, and product recovery.

Overall, the inventive coverage is directed to converting an opioid to a nor-opioid by sequential demethylation in an engineered non-plant cell: first loss of an O-linked methyl group to form a second opioid, followed by loss of an N-linked methyl group to form the nor-opioid. Dependent claims add culturing and recovery context, a metabolic pathway starting point of L-tyrosine, an engineered bacterial cell host, and a positional constraint for O-linked methyl group loss at the 3' position.

Stated Advantages

Documented Applications

No documented applications found