Compositions and methods for making (R)-Reticuline and precursors thereof
Methods that may be used for the manufacture of the chemical compound (R)-Reticuline and synthesis precursors thereof. Compositions useful for the synthesis (R)-Reticuline and synthesis precursors are also provided.
1. A method of making (R)-Reticuline or a precursor of (R)-Reticuline comprising
(a) contacting a benzylisoquinoline derivative with a microorganism expressing a heterologous enzyme capable of converting the benzylisoquinoline derivative to (R)-Reticuline or an (R)-Reticuline precursor, wherein said enzyme is a fusion protein of CYP450 and AKR and comprises an amino acid sequence that is at least 85% identical to SEQ.ID NO: 323; and
(b) growing said microorganism to produce (R)-Reticuline or a precursor of (R)-Reticuline.
2. A method for preparing (R)-Reticuline or an (R)-Reticuline precursor comprising:
(a) providing a chimeric nucleic acid sequence comprising as operably linked components:
(i) a first nucleic acid sequence encoding a CYP450 polypeptide;
(ii) a second nucleic acid sequence encoding an AKR polypeptide; and
(iii) one or more nucleic acid sequences capable of controlling expression in a host cell;
(b) introducing said chimeric nucleic acid sequence into said host cell and growing said host cell to produce CYP450 and AKR and to produce (R)-Reticuline or the (R)-Reticuline precursor, wherein said CYP450 and AKR form a fusion protein comprising an amino acid sequence that is at least 85% identical to SEQ.ID NO: 323; and
(c) recovering (R)-Reticuline or the (R)-Reticuline precursor.
3. A recombinant expression vector suitable for expression in a host cell comprising as operably linked components:
(i) a nucleic acid sequence capable of controlling expression in said host cell; and
(ii) a nucleic acid sequence encoding CYP450 and AKR,
wherein said CYP450 and AKR form a fusion protein comprising an amino acid sequence that is at least 85% identical to SEQ.ID NO: 323.
4. The method of claim 1 , wherein said method makes (R)-Reticuline.
5. The method of claim 1 , wherein said microorganism is a yeast.
6. The method of claim 1 , wherein said fusion protein of CYP450 and AKR comprises an amino acid sequence that is at least 95% identical to SEQ.ID NO: 323.
7. The method of claim 1 , further comprising converting said (R)-Reticuline or (R)-Reticuline precursor into salutaridine, salutaridine-7-O-acetate, or thebaine.
8. The method of claim 2 , wherein said host cell is a plant cell, bacterium, or yeast.
9. The method of claim 2 , wherein said method makes (R)-Reticuline.
10. The method of claim 2 , further comprising converting said (R)-Reticuline or (R)-Reticuline precursor into salutaridine, salutaridine-7-O-acetate, or thebaine.
11. The recombinant expression vector of claim 3 , wherein said host cell is a plant cell, bacterium, or yeast.
12. The method of claim 2 , wherein said fusion protein of CYP450 and AKR comprises an amino acid sequence that is at least 95% identical to SEQ.ID NO: 323.
13. The method of claim 2 , wherein said host cell is a yeast.
14. The recombinant expression vector of claim 3 , wherein said fusion protein of CYP450 and AKR comprises an amino acid sequence that is at least 95% identical to SEQ.ID NO: 323.
15. The recombinant expression vector of claim 3 , wherein said host cell is a yeast.