Engineered benzylisoquinoline alkaloid epimerases and methods of producing benzylisoquinoline alkaloids

1. A method of increasing production of an alkaloid product through the epimerization of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benyzlisoquinoline alkaloid via an engineered split epimerase in an engineered host cell in comparison to the epimerization of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benyzlisoquinoline alkaloid via a fused epimerase in similar conditions, said method comprising:

contacting said (S)-1-benzylisoquinoline alkaloid with said engineered split epimerase in said engineered host cell, thereby increasing production of an alkaloid product,

wherein contacting said (S)-1-benzylisoquinoline alkaloid with said engineered split epimerase converts said (S)-1-benzylisoquinoline alkaloid to said (R)-1-benzylisoquinoline alkaloid within said engineered host cell, wherein contacting said (S)-1-benzylisoquinoline alkaloid with said engineered split epimerase increases production of the (R)-1-benyzlisoquinoline alkaloid as compared to the epimerization of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benyzlisoquinoline alkaloid via a fused epimerase in similar conditions, and

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 separate from said reductase component of said same engineered split epimerase.

2. The method of claim 1 , wherein said fused epimerase is a wildtype epimerase.

3. The method of claim 1 , wherein said fused epimerase is a parent epimerase of said engineered split epimerase.

4. The method of claim 1 , wherein said fused epimerase is an engineered fused epimerase comprising an oxidase domain with a same sequence as said oxidase component of said engineered split epimerase.

5. The method of claim 1 , wherein said engineered split epimerase comprises one or more mutations that increase activity of said engineered split epimerase.

6. The method of claim 1 , wherein said 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.

7. The method of claim 1 , wherein a first nucleic acid which encodes said oxidase component has a first translation initiation site and wherein a second nucleic acid which encodes said reductase component has a second translation initiation site.

8. The method of claim 1 , wherein said oxidase component comprises a sequence that is encoded by a DNA sequence which comprises one or more mutations, each mutation at a codon position which corresponds to a position selected from the group consisting of positions 16, 17, 37, 50, 64, 69, 70, 110, 128, 155, 232, 234, 244, 293, 303, 328, 354, 387, 393, 427, and 477 within SEQ ID NO. 17.

9. The method of claim 1 , wherein said oxidase component comprises a sequence having at least 80% sequence identity to SEQ ID NO. 17.

10. The method of claim 1 , wherein said oxidase component comprises a sequence having at least 95% sequence identity to SEQ ID NO. 17.

11. The method of claim 1 , wherein said oxidase component comprises a sequence that is encoded by a DNA sequence which comprises one or more codon substitutions, relative to a wildtype sequence, at a codon position which corresponds to an amino acid position in SEQ ID NO. 17 selected from the group consisting of position 75, 160, 191, 256, 359, 366, and 500.

12. The method of claim 1 , wherein said reductase component comprises a sequence having at least 80% sequence identity to SEQ ID NO. 18.

13. The method of claim 1 , wherein said reductase component comprises a sequence having at least 95% sequence identity to SEQ ID NO. 18.

14. The method of claim 1 , wherein said engineered split epimerase produces at least 1% more of said (R)-1-benzylisoquinoline alkaloid than said fused epimerase under similar conditions.

15. The method of claim 1 , wherein said engineered split epimerase produces at least 5% more of said (R)-1-benzylisoquinoline alkaloid than a wildtype fused epimerase under similar conditions.

16. A method of producing an alkaloid product through the epimerization of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benyzlisoquinoline alkaloid via an engineered split epimerase in an engineered host cell said method comprising:

contacting said (S)-1-benzylisoquinoline alkaloid with said engineered split epimerase in said engineered host cell, thereby increasing production of an alkaloid product,

wherein contacting said (S)-1-benzylisoquinoline alkaloid with said engineered split epimerase converts said (S)-1-benzylisoquinoline alkaloid to said (R)-1-benzylisoquinoline alkaloid within said engineered host cell, wherein said engineered split epimerase comprises an oxidase component and a reductase component, wherein said oxidase component comprises a sequence having at least 80% sequence identity to SEQ ID NO. 17, and wherein said reductase component comprises a sequence having at least 80% sequence identity to SEQ ID NO. 18, and

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 separate from said reductase component of said same engineered split epimerase.

17. The method of claim 16 , wherein said engineered split epimerase comprises one or more mutations that increase activity of said engineered split epimerase.

18. The method of claim 16 , wherein said 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.

19. The method of claim 16 , wherein a first nucleic acid which encodes said oxidase component has a first translation initiation site and wherein a second nucleic acid which encodes said reductase component has a second translation initiation site.

20. The method of claim 16 , wherein said oxidase component comprises a sequence that is encoded by a DNA sequence which comprises one or more mutations, each mutation at a codon position which corresponds to a position selected from the group consisting of positions 16, 17, 37, 50, 64, 69, 70, 110, 128, 155, 232, 234, 244, 293, 303, 328, 354, 387, 393, 427, and 477 within SEQ ID NO. 17.

21. The method of claim 16 , wherein said oxidase component comprises a sequence having at least 85% sequence identity to SEQ ID NO. 17.

22. The method of claim 16 , wherein said oxidase component comprises a sequence having at least 95% sequence identity to SEQ ID NO. 17.

23. The method of claim 16 , wherein said oxidase component comprises a sequence that is encoded by a DNA sequence which comprises one or more codon substitutions, relative to a wildtype sequence, at a codon position which corresponds to an amino acid position in SEQ ID NO. 17 selected from the group consisting of position 75, 160, 191, 256, 359, 366, and 500.

24. The method of claim 16 , wherein said reductase component comprises a sequence having at least 85% sequence identity to SEQ ID NO. 18.

25. The method of claim 16 , wherein said reductase component comprises a sequence having at least 90% sequence identity to SEQ ID NO. 18.

26. The method of claim 16 , wherein said reductase component comprises a sequence having at least 95% sequence identity to SEQ ID NO. 18.

27. The method of claim 1 , wherein said first molecule has a net oxidase activity and wherein said second molecule has a net reductase activity.

28. The method of claim 1 , wherein the product of said first molecule comprises the substrate of said second molecule.

29. The method of claim 16 , wherein said first molecule has a net oxidase activity and wherein said second molecule has a net reductase activity.

30. The method of claim 16 , wherein the product of said first molecule comprises the substrate of said second molecule.