Ketoreductase polypeptides for the reduction of acetophenones
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 polynucleotide encoding a recombinant ketoreductase polypeptide capable of stereoselectively reducing acetophenone to (S)-1-phenethanol, which comprises an amino acid sequence that is at least 90% identical to the reference sequence of SEQ ID NO: 2, 4, or 98 and wherein said recombinant ketoreductase polypeptide has an alanine, proline, or cysteine residue at a position corresponding to position 190 of SEQ ID NO: 2, 4, or 98, wherein said the recombinant ketoreductase polypeptide comprises an amino acid sequence selected from SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, and 94, and wherein the recombinant ketoreductase polypeptide is further capable of stereoselectively reducing the substrate 2′,6′-dichloro-3′-fluoroacetophenone to the product (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol with a percent stereomeric excess of at least 99%.
2. An engineered polynucleotide encoding a recombinant ketoreductase polypeptide capable of stereoselectively reducing acetophenone to (S)-1-phenethanol, which comprises an amino acid sequence that is at least 90% identical to the reference sequence of SEQ ID NO: 2, 4, or 98 and wherein said recombinant ketoreductase polypeptide has an alanine, proline, or cysteine residue at a position corresponding to position 190 of SEQ ID NO: 2, 4, or 98, wherein said the recombinant ketoreductase polypeptide comprises an amino acid sequence selected from SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, and 94, and wherein the recombinant ketoreductase polypeptide is further capable of reducing the substrate 2′,6′-dichloro-3′-fluoracetophenone to the product (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol at a rate greater than the rate capable by the ketoreductase polypeptide having the sequence of SEQ ID NO: 6.
3. An engineered polynucleotide encoding a recombinant ketoreductase polypeptide capable of stereoselectively reducing acetophenone to (S)-1-phenethanol, which comprises an amino acid sequence that is at least 90% identical to the reference sequence of SEQ ID NO: 2, 4, or 98 and wherein said recombinant ketoreductase polypeptide has an alanine, proline, or cysteine residue at a position corresponding to position 190 of SEQ ID NO: 2, 4, or 98, wherein said the recombinant ketoreductase polypeptide comprises an amino acid sequence selected from SEQ ID NO: 8, 10, 14, 16, 18, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, and 94, and wherein the recombinant ketoreductase polypeptide is further capable of reducing the substrate 2′,6′-dichloro-3′-fluoracetophenone to the product (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol at a rate that is at least 450% greater than the rate capable by the ketoreductase polypeptide having the sequence of SEQ ID NO: 6.
4. An engineered polynucleotide encoding a recombinant ketoreductase polypeptide capable of stereoselectively reducing acetophenone to (S)-1-phenethanol, which comprises an amino acid sequence that is at least 90% identical to the reference sequence of SEQ ID NO: 2, 4, or 98 and wherein said recombinant ketoreductase polypeptide has an alanine, proline, or cysteine residue at a position corresponding to position 190 of SEQ ID NO: 2, 4, or 98, wherein said the recombinant ketoreductase polypeptide comprises an amino acid sequence selected from SEQ ID NO: 18, 32, 34, 36, 38, 40, 42, 44, 46, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, and 94, and wherein the recombinant ketoreductase polypeptide is further capable of reducing the substrate 2′,6′-dichloro-3′-fluoracetophenone to the product (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol at a rate that is at least 1500% greater than the rate capable by the ketoreductase polypeptide having the sequence of SEQ ID NO: 6.
5. An expression vector comprising the engineered polynucleotide of claim 1 .
6. The vector of claim 5 , wherein said engineered polynucleotide is operably linked with regulatory sequences suitable for expression of said engineered polynucleotide in a suitable host cell.
7. The vector of claim 6 , wherein said host cell is a prokaryotic or eukaryotic cell.
8. The vector of claim 7 , wherein said host cell is a prokaryotic cell.
9. The vector of claim 7 , wherein said host cell is E. coli.
10. A host cell comprising the vector of claim 5 .
11. A method for producing a recombinant ketoreductase polypeptide comprising culturing the host cell of claim 10 under conditions such that a recombinant ketoreductase polypeptide is produced.
12. The method of claim 11 , further comprising the step of recovering said recombinant ketoreductase polypeptide.