Enzymatic synthesis of kavalactones and flavokavains
View Patent ↗Disclosed are methods, compositions, proteins, nucleic acids, cells, vectors, compounds, reagents, and systems for the preparation of kavalactones, flavokavains, and kavalactone and flavokavain biosynthetic intermediates using enzymes expressed in heterologous host cells, such as microorganisms or plants, or using in vitro enzymatic reactions. This invention also provides for the expression of the enzymes by recombinant cell lines and vectors. Furthermore, the enzymes can be components of constructs such as fusion proteins. The kavalactones produced can be utilized to treat anxiety disorder, insomnia, and other psychological and neurological disorders. The flavokavains produced can be utilized to treat various cancers including colon, bladder, and breast cancers.
1. A method for producing a compound of Formula (III), the method comprising:
(i) reacting a compound of Formula (II), or a salt thereof, with malonyl-CoA using an enzyme that has an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of PmSPS 1 polypeptide of SEQ ID NO: 2 or an enzyme that has an amino acid sequence that is at least 90% sequence identical to the amino acid sequence of PmSPS2 polypeptide of SEQ ID NO: 3; and
(ii) isolating a compound of Formula (III):
wherein:
is a single bond or a double bond;
each of R 1 , R 2 , R 3 , R 6 , and R 7 independently is hydrogen, optionally substituted, cyclic or acyclic aliphatic, or OR x , wherein R x is hydrogen or optionally substituted, cyclic or acyclic aliphatic; and
each of R 4 and R 5 independently is hydrogen or optionally substituted, cyclic or acyclic aliphatic.
2. The method of claim 1 , wherein R 1 is selected from the group consisting of hydrogen, —OH, and —OCH 3 .
3. The method of claim 1 , wherein R 2 is selected from the group consisting of hydrogen, —OH, and —OCH 3 .
4. The method of claim 1 , wherein R 3 is selected from the group consisting of hydrogen, —OH, and —OCH 3 .
5. The method of claim 1 , wherein R 4 and R 5 are hydrogen.
6. The method of claim 1 , wherein R 6 is selected from the group consisting of hydrogen, —OH, and —OCH 3 .
7. The method of claim 1 , wherein R 7 is selected from the group consisting of hydrogen, —OH, and —OCH 3 .
8. The method of claim 1 , wherein R 6 is —OH.
9. The method of claim 1 , wherein R 7 is hydrogen.
10. The method of claim 1 , wherein R 1 , R 2 , and R 3 are hydrogen.
11. The method of claim 1 , wherein R 1 , R 2 , and R 3 are —OH.
12. The method of claim 1 , wherein R 1 and R 3 are —OH.
13. The method of claim 1 , wherein R 2 and R 3 are —OH.
14. The method of claim 1 , wherein R 2 is —OCH 3 .
15. The method of claim 1 , wherein the enzyme is produced by a recombinant cell line.
16. The method of claim 1 , wherein the enzyme is obtained from a non-human organism.
17. The method of claim 1 , wherein the enzyme is in a cell.
18. The method of claim 17 , wherein the enzyme is heterologous to the cell.
19. The method of claim 1 , wherein the enzyme has an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of PmSPS1 polypeptide of SEQ ID NO: 2 or an enzyme that has an amino acid sequence that is at least 95% sequence identical to the amino acid sequence of PmSPS2 polypeptide of SEQ ID NO: 3.
20. The method of claim 1 , wherein the enzyme comprises PmSPS1 polypeptide of SEQ ID NO: 2 or PmSPS2 polypeptide of SEQ ID NO: 3.
21. The method of claim 17 , wherein the cell is a bacterial cell.
22. The method of claim 17 , wherein the cell is a yeast cell.