Engineered benzylisoquinoline alkaloid epimerases and methods of producing benzylisoquinoline alkaloids
Inventors
Smolke, Christina D. • Wells, Derek H.
Assignees
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Publication Number
US-11427827-B2
Publication Date
2022-08-30
Expiration Date
Abstract
The present disclosure provides systems and methods for increasing production of an alkaloid product through the epimerization of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benyzlisoquinoline alkaloid via an engineered epimerase in an engineered host cell. A (S)-1-benzylisoquinoline alkaloid is contacted with said engineered epimerase. Contacting said (S)-1-benzylisoquinoline alkaloid with said engineered epimerase converts said (S)-1-benzylisoquinoline alkaloid to said (R)-1-benzylisoquinoline alkaloid.
Core Innovation
The invention describes an engineered host cell comprising an engineered split epimerase that increases conversion of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benzylisoquinoline alkaloid relative to conversion via a fused epimerase under similar conditions. The increase is measured by a quantity of (R)-1-benzylisoquinoline alkaloid produced within the engineered host cell.
The engineered split epimerase comprises an oxidase component and a reductase component, where the oxidase component is expressed as a separate polypeptide from the reductase component. The oxidase component has the same sequence as an oxidase component of the fused epimerase, and the reductase component has the same sequence as a reductase component of the fused epimerase.
Conversion is selected from (S)-Reticuline to (R)-Reticuline and (S)-N-methylcoclaurine to (R)-N-methylcoclaurine, with the oxidase component comprising a sequence that shares at least 80% sequence identity with SEQ ID NO. 17 and the reductase component comprising a sequence that shares at least 80% sequence identity with SEQ ID NO. 18. The document further includes variant sets with specific amino-acid substitutions and sequence-identity markers at defined positions relative to SEQ ID NO. 17 and SEQ ID NO. 18.
Claims Coverage
The document provides two independent claims. Both define an engineered split epimerase that increases conversion of a (S)-1-benzylisoquinoline alkaloid to the corresponding (R)-1-benzylisoquinoline alkaloid relative to conversion via a fused epimerase under similar conditions, while specifying separate oxidase and reductase polypeptides with sequence identity to SEQ ID NO. 17 and SEQ ID NO. 18.
Engineered split epimerase in an engineered host cell for increased (S)-to-(R) benzylisoquinoline alkaloid conversion
An engineered host cell comprising an engineered split epimerase that increases conversion of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benzylisoquinoline alkaloid relative to conversion via a fused epimerase under similar conditions, where conversion is measured by a quantity of (R)-1-benzylisoquinoline alkaloid produced within the engineered host cell.
Engineered split epimerase with oxidase/reductase separation and specified sequence identity
An engineered split epimerase that increases conversion of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benzylisoquinoline alkaloid relative to conversion via a fused epimerase under similar conditions, where the engineered split epimerase comprises an oxidase component and a reductase component expressed as separate polypeptides, with the oxidase component sharing at least 80% sequence identity with SEQ ID NO. 17 and the reductase component sharing at least 80% sequence identity with SEQ ID NO. 18.
Across the independent claims, the core inventive concept is an engineered split epimerase with an oxidase component and a reductase component expressed as separate polypeptides, having sequence identity constraints to SEQ ID NO. 17 and SEQ ID NO. 18, configured to increase conversion of (S)-Reticuline or (S)-N-methylcoclaurine to the corresponding (R)-products relative to a fused epimerase under similar conditions.
Stated Advantages
Increases conversion of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benzylisoquinoline alkaloid relative to conversion via a fused epimerase under similar conditions.
Improved production performance and chiral (R)-reticuline enrichment.
Documented Applications
Producing plant specialized metabolites and benzylisoquinoline alkaloids.
Producing drug libraries.
Enabling plant enzyme discovery.
Producing thebaine and morphinan, nor-opioid, and nal-opioid alkaloids.
Production of a (R)-1-benzylisoquinoline alkaloid from a (S)-1-benzylisoquinoline alkaloid using an engineered host cell.
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