Ketoreductase polypeptides and related polynucleotides
AssigneeCodexis, Inc.
InventorsS. Davis, Gjalt Huisman, Stephane Jenne, Anke Krebber, Lisa Newman
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Abstract
The present invention is directed to variant polypeptides having enhanced ketoreductase activity and/or thermostability for use in the stereospecific reduction of ketones. In addition, the present invention is directed to polynucleotides that encode the ketoreductase polypeptides, including codon optimized versions of the polynucleotides which provide for enhanced expression in host cells. In another aspect, the present invention is directed to nucleotide constructs, vectors and host cells that are transformed with polynucleotides of the present invention.
Claims15
A KRED polypeptide having at least 1.5 times the KRED activity of the polypeptide of SEQ ID NO:
- 2, and being at least 90% homologous with the amino acid sequence of SEQ ID NO:
- 2.
A KRED polypeptide having 1.5 to about 25 times the KRED activity of the polypeptide of SEQ ID NO:
- 2, but having the backbone of SEQ ID NO:
- 2 and from one to twenty residue changes selected from the group consisting of A2V;
- K3E;
- F5L or C;
- N7K;
- E9G or K;
- A12V;
- P13L;
- P14A;
- A16G or V;
- T18A;
- K19I;
- N20D or S;
- E21K;
- S22N or T;
- Q24H or R;
- V25A;
- N32S or D;
- A36T;
- S41G;
- S42N;
- 145L;
- A48T;
- V56A;
- V60I;
- Y64H;
- N65K, D, Y or S;
- S66G or R;
- H67L or Q;
- D68G or N;
- G71D;
- E74K or G;
- K78R;
- K79R;
- K85R;
- A86V;
- N90D;
- S93Nor C;
- D95N, G, V, Y or E;
- K98R;
- Q99L, R, or H;
- T100A;
- I101V;
- Q103R;
- I105V or T;
- K106R or Q;
- H110Y, C or R;
- V114A;
- A116G;
- I120V;
- K124R;
- D129G or N;
- D131G or V;
- D132N;
- K134M, V, E or R;
- D137N or G;
- Q138L;
- V140I;
- D143N;
- L144F;
- K145R;
- V147A;
- V150A;
- H153Y or Q;
- H157Y;
- F158L or Y;
- R159K;
- E160G or V;
- F162Y or S;
- E163G or K;
- E165D, G or K;
- K1671 or R;
- A170S;
- V172I;
- F173C;
- M177V or T;
- H180Y;
- V184I;
- T190A;
- A193V;
- A194V;
- F201L;
- K203R;
- F209Y;
- V218I;
- N224S;
- E226K, G or D;
- S228T;
- D229A;
- V231I or A;
- Q233K or R;
- E234G or D;
- T235K or A;
- N237Y;
- K238R or E;
- T251A;
- V255A;
- F260L;
- A262V;
- T272A;
- 1274L;
- 1275L or V;
- and P283R.
A KRED polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:
- 224, 244, 246, 250, 252, 254, 256, 260, 304, 344, 354, 358, 360, 364, 368, 374, 382, 386, 388, 400, 408, 438, 440, 448, 470, 484, 486, 488, 490, 502, 506, 508, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, and 542.
A KRED polypeptide having 1.5 to about 25 times the ketoreductase activity of the polypeptide of SEQ ID NO:
- 2, and either
- (a) having an amino acid sequence which has at least 90% homology with SEQ ID NO:
- 224, 244, 246, 250, 252, 254, 256, 260, 304, 344, 354, 358, 360, 364, 368, 374, 382, 386, 388, 400, 408, 438, 440, 448, 470, 484, 486, 488, 490, 502, 506, 508, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, and 542;
- (b) is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with either (i) the nucleotide sequence of SEQ ID NO:
- 223, 243, 245, 249, 251, 253, 255, 259, 303, 343, 353, 357, 359, 363, 367, 373, 381, 385, 387, 399, 407, 437, 439, 447, 469, 483, 485, 487, 489, 501, 505, 507, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, or 541, (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i) or (ii);
- (c) is a variant of the polypeptide of SEQ ID NO:
- 224, 244, 246, 250, 252, 254, 256, 260, 303, 344, 354, 358, 360, 364, 368, 374, 382, 386, 388, 400, 408, 438, 440, 448, 470, 484, 486, 488, 490, 502, 506, 508, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, or 542 comprising a substitution, deletion, and/or insertion of one to six amino acids;
- (d) is a fragment of at least 220 amino acid residues from a polypeptide of SEQ ID NO:
- 224, 244, 246, 250, 252, 254, 256, 260, 303, 344, 354, 358, 360, 364, 368, 374, 382, 386, 388, 400, 408, 438, 440, 448, 470, 484, 486, 488, 490, 502, 506, 508, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, or 542;
- or
- (e) is a polypeptide of (a), (b), (c) or (d) that retains more than 60% of the initial KRED activity after incubation at 50° C., pH 7 for 60 minutes.
Dependencies are inferred from the claim text.