Methods of producing epimerases and benzylisoquinoline alkaloids
A method of epimerizing an (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid is provided. The method comprises contacting the (S)-1-benzylisoquinoline alkaloid with at least one enzyme. Contacting the (S)-1-benzylisoquinoline alkaloid with the at least one enzyme converts the (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid.
1. A method of producing an (R)-1-benzylisoquinoline alkaloid, the method comprising:
(a) culturing an engineered non-plant cell in a cell culture comprising a feedstock, wherein the engineered non-plant cell stably expresses a plurality of heterologous enzymes within the same engineered non-plant-cell for the production of an (S)-1-benzylisoquinoline alkaloid wherein the plurality of heterologous enzymes comprise tyrosine hydroxylase (TyrH), L-DOPA decarboxylase (DODC), norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), cytochrome P450 80B1 (CYP80B1), and 4′-O-methyltransferase (4′OMT),
(b) producing an (S)-1-benzylisoquinoline alkaloid within the same engineered non-plant cell,
(c) contacting, within the same engineered non-plant cell, the (S)-1-benzylisoquinoline alkaloid that is produced within the engineered non-plant cell with at least one heterologous, fused enzyme expressed within the same engineered non-plant cell that converts the (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid within the engineered non-plant cell, and
(d) producing the (R)-1-benzylisoquinoline alkaloid within the engineered non-plant cell.
2. The method of claim 1 , wherein the at least one enzyme that converts the (S)-1-benzylisoquinoline alkaloid to the (R)-1-benzylisoquinoline alkaloid is encoded by at least one coding sequence within a chromosome of the engineered non-plant cell.
3. The method of claim 2 , further comprising: adding an (S)-1-benzylisoquinoline alkaloid to the cell culture.
4. The method of claim 2 , further comprising: recovering the (R)-1-benzylisoquinoline alkaloid, or a derivative thereof, from the cell culture.
5. The method of claim 1 , wherein the at least one enzyme that converts the (S)-1-benzylisoquinoline alkaloid to the (R)-1-benzylisoquinoline alkaloid comprises an epimerase.
6. The method of claim 5 , wherein the epimerase comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.
7. The method of claim 5 , wherein the epimerase comprises an oxidase domain and a reductase domain.
8. The method of claim 7 , wherein the oxidase domain is a cytochrome P450 oxidase-like domain.
9. The method of claim 7 , wherein the reductase domain is a codeinone reductase-like domain.
10. The method of claim 1 , wherein the engineered non-plant cell is an engineered yeast cell or an engineered bacteria cell.
11. The method of claim 1 , wherein the (S)-1-benzylisoquinoline alkaloid is (S)-reticuline.
12. The method of claim 1 , wherein the (S)-1-benzylisoquinoline alkaloid is produced within the engineered non-plant cell by a metabolic pathway starting with L-tyrosine.
13. The method of claim 1 , wherein the at least one enzyme that converts the (S)-1-benzylisoquinoline alkaloid to the (R)-1-benzylisoquinoline alkaloid comprises a first enzyme having an oxidase domain and a second enzyme having a reductase domain, wherein the first enzyme is separate from the second enzyme.
14. The method of claim 5 , wherein the epimerase comprises an amino acid sequence having at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.
15. The method of claim 5 , wherein the epimerase comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.
16. The method of claim 5 , wherein the epimerase comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.
17. The method of claim 5 , wherein the activity of the epimerase is encoded in a single polypeptide.
18. The method of claim 5 , wherein the activity of the epimerase is encoded across multiple polypeptides.
19. The method of claim 18 , wherein the activity of the epimerase is encoded across two polypeptides.
20. A method of producing an (R)-1-benzylisoquinoline alkaloid, the method comprising:
(a) culturing an engineered non-plant cell in a cell culture comprising a feedstock, wherein the engineered non-plant cell stably expresses a plurality of heterologous enzymes within the same engineered non-plant-cell for the production of an (S)-1-benzylisoquinoline alkaloid wherein the plurality of heterologous enzymes comprise tyrosine hydroxylase (TyrH), L-DOPA decarboxylase (DODC), monoamine oxidase (MAO), norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), and 4′-O-methyltransferase (4′OMT),
(b) producing an (S)-1-benzylisoquinoline alkaloid within the same engineered non-plant cell,
(c) contacting, within the same engineered non-plant cell, the (S)-1-benzylisoquinoline alkaloid that is produced within the engineered non-plant cell with at least one heterologous, fused enzyme expressed within the same engineered non-plant cell that converts the (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid within the engineered non-plant cell, and
(d) producing the (R)-1-benzylisoquinoline alkaloid within the engineered non-plant cell.
21. The method of claim 5 , wherein the epimerase comprises an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.
22. The method of claim 5 , wherein the epimerase comprises the amino acid sequence of SEQ ID NO: 8.
23. The method of claim 20 , wherein the epimerase comprises an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.
24. The method of claim 20 , wherein the epimerase comprises the amino acid sequence of SEQ ID NO: 8.