Ketoreductase polypeptides for the production of azetidinone
View Patent ↗The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize a variety of chiral compounds.
1. An engineered polypeptide having ketoreductase activity, wherein the ketoreductase comprises an amino acid sequence that is at least 90% identical to a reference sequence of SEQ ID NO: 8 and comprises the amino acid substitution: F147M.
2. The engineered ketoreductase polypeptide of claim 1 , wherein the amino acid sequence further comprises the amino acid substitutions Y190H or P; V196L; I226V; and Y249H, R, or W.
3. The engineered ketoreductase polypeptide of claim 2 , wherein the amino acid sequence further comprises one or more amino acid substitutions selected from the group consisting of: H40R; A94E, F, or G; S96V; and E145D or F.
4. The engineered ketoreductase polypeptide of claim 3 , wherein the amino acid sequence comprises the amino acid substitutions: A94G, A96V, E145F, F147M, Y190P, V196L, I226V, and Y249W.
5. The engineered ketoreductase polypeptide of claim 3 , wherein the amino acid sequence comprises the amino acid substitutions: A94G, A96V, E145F, F147M, Y190P, V196L, I226V, and Y249R.
6. The engineered ketoreductase polypeptide of claim 3 , wherein the amino acid sequence comprises the amino acid substitutions: A94F, A96V, E145F, F147M, Y190P, V196L, I226V, and Y249W.
7. The engineered ketoreductase polypeptide of claim 3 , wherein the amino acid sequence comprises the amino acid substitutions: A94F, A96V, E145F, F147M, Y190P, V196L, I226V, and Y249H.
8. The engineered ketoreductase polypeptide of claim 3 , wherein the amino acid sequence comprises the amino acid substitutions: A94F, A96V, E145F, F147M, Y190H, V196L, 1226V, and Y249H.
9. The engineered ketoreductase polypeptide of claim 3 , wherein the amino acid sequence comprises the amino acid substitutions: A94F, A96V, F147M, and Y190P.
10. The engineered ketoreductase polypeptide of claim 3 , wherein the amino acid sequence has additionally 1-15 substitutions at other amino acid residue positions as compared to the reference sequence.
11. The engineered ketoreductase polypeptide of claim 3 , wherein the additional 1-15 substitutions are conservative mutations.
12. The engineered ketoreductase polypeptide of claim 1 in which the ketoreductase is capable of converting the substrate, methyl-2-benzamidomethyl-3-oxobutyrate, to the product, 2R,3R-methyl-2-benzamidomethyl-3-hydroxybutyrate.
13. The engineered ketoreductase polypeptide of claim 12 , wherein in which the polypeptide is capable of converting the substrate, methyl-2-benzamidomethyl-3-oxobutyrate, to the product, 2R,3R-methyl-2-benzamidomethyl-3-hydroxybutyrate in stereomeric excess.