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 - 57 . (canceled)
58 . A method for producing bakuchiol, the method comprising
growing a microbial cell expressing a bakuchiol synthase 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; and
extracting bakuchiol from the microbial cell.
59 . The method of claim 58 , wherein the microbial cell is Escherichia coli or yeast.
60 . The method of claim 58 , wherein the microbial cell is Escherichia coli or Saccharomyces cerevisiae.
61 . The method of claim 58 , wherein the bakuchiol synthase comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3.
62 . The method of claim 58 , wherein the bakuchiol synthase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3.
63 . The method of claim 58 , wherein the bakuchiol synthase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3.
64 . The method of claim 58 , wherein the bakuchiol synthase 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.
65 . The method of claim 58 , wherein the bakuchiol synthase comprises an amino acid sequence selected from SEQ ID NOs: 67 to 81.
66 . The method of claim 58 , wherein the bakuchiol synthase comprises V48S and W209C substitutions, wherein the residue position is relative to SEQ ID NO: 3.
67 . The method of claim 58 , wherein the bakuchiol synthase comprises T65C and W209C substitutions, wherein the residue position is relative to SEQ ID NO: 3.
68 . The method of claim 58 , wherein the bakuchiol synthase comprises a substitution combination of E54F, W209C, D279K, M287V, V312L, F318R, and S342G, wherein the residue position is relative to SEQ ID NO: 3.
69 . The method of claim 58 , wherein the bakuchiol synthase comprises a substitution combination of P205L, L206Y, W209Y, M287V, V312L, and S342G, wherein the residue position is relative to SEQ ID NO: 3.
70 . The method of claim 58 , wherein the microbial cell is cultured in a medium comprising p-coumaric acid.
71 . A method for producing bakuchiol, the method comprising:
growing 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; and
extracting bakuchiol from the microbial cell.
72 . The method of claim 71 , wherein the microbial cell is Escherichia coli or yeast.
73 . The method of claim 72 , wherein the microbial cell is cultured in a medium comprising p-coumaric acid.
74 . A method for producing bakuchiol, the method comprising:
growing a microbial cell expressing a bakuchiol synthase and capable of producing bakuchiol; and
extracting bakuchiol from the microbial cell.
75 . The method of claim 74 , wherein the microbial cell is Escherichia coli or yeast.
76 . The method of claim 75 , wherein the microbial cell is cultured in a medium comprising p-coumaric acid.
77 . The method of claim 74 , wherein the bakuchiol synthase is from a plant species selected from the group consisting of Psoralea corylifolia, P. grandulosa, P. drupaceae, Ulmus davidiana, Otholobium pubescens, Piper longum , and Aerva sangulnolenta Blum.