Engineered enzymes and bioproduction of bakuchiol
The present disclosure relates to synthetic biology and, in particular, the bioproduction of bakuchiol, and engineered enzymes for producing the same.
1. A polypeptide comprising (a) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3; and (b) a N-terminus truncation of at least one amino acid but not more than 100 amino acids relative to SEQ ID NO: 2.
2. A nucleic acid molecule encoding the polypeptide of claim 1 .
3. A cell expressing the polypeptide of claim 1 .
4. The cell of claim 3 , wherein the cell is a microbial cell.
5. A microbial cell expressing a bakuchiol synthase and capable of producing bakuchiol, wherein the bakuchiol synthase is (i) BAK28 or a variant thereof, or (ii) BAK36 or a variant thereof.
6. The microbial cell of claim 5 , wherein the microbial cell is E. coli or yeast.
7. The microbial cell of claim 5 , wherein the microbial cell is E. coli or Saccharomyces cerevisiae.
8. The nucleic acid molecule of claim 2 , wherein the polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3.
9. The nucleic acid molecule of claim 2 , wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3.
10. The nucleic acid molecule of claim 2 , wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3.
11. The nucleic acid molecule of claim 2 , wherein the polypeptide comprises an amino acid substitution relative to SEQ ID NO: 3 at a site corresponding to a residue position selected from W209 and V312 of SEQ ID NO: 3.
12. The nucleic acid molecule of claim 2 , wherein the polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 67 to 81.
13. The cell of claim 3 , wherein the polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3.
14. The cell of claim 3 , wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3.
15. The cell of claim 3 , wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3.
16. The cell of claim 3 , wherein the polypeptide comprises an amino acid substitution relative to SEQ ID NO: 3 at a site corresponding to a residue position selected from W209 and V312 of SEQ ID NO: 3.
17. The cell of claim 3 , wherein the polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 67 to 81.
18. The cell of claim 3 , wherein the polypeptide comprises V48S and W209C substitutions, wherein the residue position is relative to SEQ ID NO: 3.
19. The cell of claim 3 , wherein the polypeptide comprises T65C and W209C substitutions, wherein the residue position is relative to SEQ ID NO: 3.
20. The cell of claim 3 , wherein the polypeptide comprises a substitution combination of E54F, W209C, D279K, M287V, V312L, F318R, and S342G, wherein the residue position is relative to SEQ ID NO: 3.
21. The cell of claim 3 , wherein the polypeptide comprises a substitution combination of P205L, L206Y, W209Y, M287V, V312L, and S342G, wherein the residue position is relative to SEQ ID NO: 3.