Ketoreductase polypeptides and polynucleotides
The present invention provides engineered ketoreductase and phosphite dehydrogenase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase and phosphite dehydrogenase enzymes, as well as polynucleotides encoding the engineered ketoreductase and phosphite dehydrogenase enzymes, host cells capable of expressing the engineered ketoreductase and phosphite dehydrogenase enzymes, and methods of using the engineered ketoreductase and phosphite dehydrogenase enzymes to synthesize a chiral catalyst used in the synthesis of antiviral compounds, such as nucleoside inhibitors. The present invention further provides methods of using the engineered enzymes to deracemize a chiral alcohol in a one-pot, multi-enzyme system.
1. An engineered ketoreductase variant having at least 95% sequence identity to SEQ ID NO: 2, wherein said engineered ketoreductase variant comprises a substitution in the amino acid sequence at position 57, as compared to SEQ ID NO: 2.
2. The engineered ketoreductase variant of claim 1 having at least 95% sequence identity to SEQ ID NO:2, and further comprising at least one substitution or substitution set at one or more positions selected from positions 37, 37/211, 37/211/229, 37/229, 45, 52, 52/110/272/296, 52/272, 52/272/274/279/296, 52/272/279/296, 55/276, 56, 104/114, 104/114/229, 286, 79/83/275/276, 83, 83/275/276, 83/276, 104, 110, 114, 138/146/258/289, 211, 211/229, 228, 229, 263, 268, 272, 274, 275/276, 276, 279, and 309, wherein said positions are numbered with reference to SEQ ID NO:2.
3. The engineered ketoreductase variant of claim 1 , comprising a polypeptide sequence having at least 97% sequence identity to SEQ ID NO: 2.
4. The engineered ketoreductase variant of claim 1 , comprising a polypeptide sequence having at least 99% sequence identity to SEQ ID NO: 2.
5. The engineered ketoreductase variant of claim 1 , wherein said engineered ketoreductase comprises a polypeptide sequence selected from the even-numbered sequences set forth in SEQ ID NOS: 6, 16, 36, 96, 100-110.
6. A composition comprising at least one engineered ketoreductase variant provided in claim 1 .