Engineered benzylisoquinoline alkaloid epimerases and methods of producing benzylisoquinoline alkaloids
AssigneeAntheia, Inc.
InventorsChristina D. Smolke, Derek H. Wells
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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.
Claims14
An engineered host cell which comprises an engineered split epimerase, wherein said engineered split epimerase increases conversion of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benzylisoquinoline alkaloid relative to conversion of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benzylisoquinoline alkaloid, as measured by a quantity of (R)-1-benzylisoquinoline alkaloid produced within the engineered host cell, via a fused epimerase under similar conditions, wherein said engineered split epimerase comprises an oxidase component and a reductase component, wherein said oxidase component of said engineered split epimerase comprises a first molecule, and a reductase component of said same engineered split epimerase comprises a second molecule, and wherein said oxidase component of said engineered split epimerase is expressed as a separate polypeptide from said reductase component of said same engineered split epimerase, and further wherein the oxidase component of the split epimerase has the same sequence as an oxidase component of the fused epimerase, and wherein the reductase component of the split epimerase has the same sequence as a reductase component of the fused epimerase, and further wherein the conversion of the (S)-1-benzylisoquinoline alkaloid to the (R)-1-benzylisoquinoline alkaloid is selected from the group consisting of:
- (S)-Reticuline to (R)-Reticuline, and
- (S)-N-methylcoclaurine to (R)-N-methylcoclaurine, and
- wherein said oxidase component comprises a sequence that shares at least 80% sequence identity with SEQ ID NO. 17, and
- wherein said reductase component comprises a sequence that shares at least 80% sequence identity with SEQ ID NO. 18.
An engineered split epimerase that increases conversion of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benzylisoquinoline alkaloid relative to conversion of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benzylisoquinoline alkaloid via a fused epimerase under similar conditions, wherein the engineered split epimerase comprises one or more mutations selected from the group consisting of a localization mutation, a cytochrome P450 reductase interaction mutation, and an accessibility mutation, wherein said engineered split epimerase comprises an oxidase component and a reductase component, wherein said oxidase component of said engineered split epimerase comprises a first molecule, and a reductase component of said same engineered split epimerase comprises a second molecule, and wherein said oxidase component of said engineered split epimerase is expressed as a separate polypeptide from said reductase component of said same engineered split epimerase, and further wherein the conversion of the (S)-1-benzylisoquinoline alkaloid to the (R)-1-benzylisoquinoline alkaloid is selected from the group consisting of:
- (S)-Reticuline to (R)-Reticuline, and
- (S)-N-methylcoclaurine to (R)-N-methylcoclaurine, and
- wherein said oxidase component comprises a sequence that shares at least 80% sequence identity with SEQ ID NO. 17, and
- wherein said reductase component comprises a sequence that shares at least 80% sequence identity with SEQ ID NO. 18.
Dependencies are inferred from the claim text.