# US 2010/0167345 A1 · Halohydrin dehalogenases and related polynucleotides

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- Number: US 2010/0167345 A1
- Document ID: US20100167345A1
- Status: Published application
- Kind: A1
- Application number: 12/573,824
- Filed: 2009-10-05
- Published: 2010-07-01
- Earliest claimed: 2003-08-11
- Assignees: Codexis, Inc.
- Inventors: S. Christopher Davis; Richard John Fox; Gjalt W. Huisman; Vesna Gavrilovic; Lisa Marie Newman
- Web page: https://patentgrep.com/patent/US20100167345A1

## CPC

- C12N 9/88: Lyases (4.)
- C12N 9/14: Hydrolases (3)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to novel halohydrin dehalogenase polypeptides and the polynucleotides that encode them. These polypeptides are useful in the production of 4-substituted-3-butyric acid derivatives and vicinal cyano, hydroxyl substituted carboxylic acid esters. The invention also provides related vectors, host cells and methods.

## Claims

### Claim 1

An isolated polypeptide having HHDH activity, wherein the polypeptide comprises an amino acid sequence consisting of
(a) an amino acid sequence that is at least 99% identical to SEQ ID NO: 4, 12, 16, 18, 34, 38, 44, 48, 52, 66, 80, 84, 114, 154, 158, 170, or 270;
(b) a polypeptide having an amino acid sequence that is at least 98% identical to SEQ ID NO: 10, 14, or 68, 118, 164, 166, or 180;
(c) an amino acid sequence that is at least 97% identical to SEQ ID NO: 110, 162, 262, 422, 440 or 520;
(d) an amino acid sequence that is at least 96% identical to SEQ ID NO: 116 or 448;
(e) an amino acid sequence that is at least 95% identical to SEQ ID NO: 264, 266, 470 or 476;
(f) an amino acid sequence that is at least 93% identical to SEQ ID NO: 200;
(g) an amino acid sequence that is at least 89% identical to SEQ ID NO: 442;
(h) an amino acid sequence that is at least 88% identical to SEQ ID NO: 702;
(i) an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, when optimally aligned with SEQ ID NO: 2, and which comprises at least one amino acid residue selected from the group consisting of T at (residue) position 2, A or P or S at position 3, V at position 4, D at position 6, either I or F at position 9, L at position 10, S at position 13, S at position 14, K at position 15, C at position 16, T or R at position 17, either C or S or K at position 20, T at position 24, Q at position 26, F at position 28, T at position 29, A at position 30, L at position 31, G at position 33, R at position 34, L at position 35, N at position 36, H at position 37, D at position 40, L at position 44, P at position 45, either P or A at position 47, N at position 52, V at position 54, R at position 55, D at position 56, K at position 58, G or D at position 61, Vat position 63, R at position 72, I at position 75, P at position 76, C at position 78, Y at position 82, either S or L at position 84, A at position 85, E at position 91, D at position 93, Q or G at position 95, N at position 96, K at position 107, A at position 112, either T, S or G at position 114, A at position 115, P at position 117, N at position 120, E at position 121, P at position 122, R at position 126, V at position 130, S at position 133, A or V at position 134, L, W or V at position 136, H at position 139, I or R at position 142, S at position 144, S at position 146, T at position 152, S at position 153, either S or A at position 154, V at position 168, T at position 169, F at position 177, V at position 178, I at position 180, G or I at position 181, K at position 184, Y at position 186, L at position 194, N at position 198, M at position 199, E at position 215, G at position 236, Vat position 237, L at position 238, T at position 240, either I or A or V at position 245, Y at position 249, V or I at position 252, and V at position 254; or
(j) an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleic acid corresponding to SEQ ID NO: 1, and wherein the encoded polypeptide, when optimally aligned with SEQ ID NO: 2, comprises an amino acid sequence having at least one amino acid residue selected from the group consisting of T at (residue) position 2, A or P or S at position 3, V at position 4, D at position 6, either I or F at position 9, L at position 10, S at position 13, S at position 14, K at position 15, C at position 16, T or R at position 17, either C or S or K at position 20, T at position 24, Q at position 26, F at position 28, T at position 29, A at position 30, L at position 31, G at position 33, R at position 34, L at position 35, N at position 36, H at position 37, D at position 40, L at position 44, P at position 45, either P or A at position 47, N at position 52, V at position 54, R at position 55, D at position 56, K at position 58, G or D at position 61, V at position 63, R at position 72, I at position 75, P at position 76, C at position 78, Y at position 82, either S or L at position 84, A at position 85, E at position 91, D at position 93, Q or G at position 95, N at position 96, K at position 107, A at position 112, either T, S or G at position 114, A at position 115, P at position 117, N at position 120, E at position 121, P at position 122, R at position 126, V at position 130, S at position 133, A or V at position 134, L, W or V at position 136, H at position 139, I or R at position 142, S at position 144, S at position 146, T at position 152, S at position 153, either S or A at position 154, V at position 168, T at position 169, F at position 177, V at position 178, I at position 180, G or I at position 181, K at position 184, Y at position 186, L at position 194, N at position 198, M at position 199, Eat position 215, G at position 236, V at position 237, L at position 238, T at position 240, either I or A or V at position 245, Y at position 249, V or I at position 252, and V at position 254.

### Claim 2 (depends on 1)

The isolated polypeptide of claim 1, wherein the polypeptide has an amino acid sequence corresponding to SEQ ID NO: 2, but with one or more substitutions selected from the group consisting of: S2T, either T3A or T3P, A4V, V6D, either V9I or V9F, K10L, G13S, G14S, M15K, G16C, either S17T or S17R, either R20S, R20C or R2OK, A24T, H26Q, V28F, A29T, C30A, H31L, E33G, S34R, F35L, K36N, Q37H, E40D, F44L, A45P, either T47P or T47A, K52N, M54V, S55R, E56D, E58K, either E61G or E61D, I63V, Q72R, V75I, L76P, S78C, F82Y, either P84S or P84L, E85Q, K91E, A93D, E95Q or E95G, D96N, R107K, V112A, either A114T or A114G or A114S, V115A, S117P, K120N, K121E, R122P, H126R, 1130V, A133S, T134A or T134V, F136L or F136W or F136V, W139H, L1421 or L142R, T144S, T146S, A152T, C153S, either T154S or T154A, 1168V, P169T, Y177F, L178V, S1801, E181G or E181I, P184K, F186Y, T1941, H198N, V199M, K215E, V236G, F237V, W238L, A240T, either M245I or M245A or M245V, W249Y, M252V or M252I, and E254V.

### Claim 3 (depends on 1)

The isolated polypeptide of claim 1, wherein the polypeptide has at least 1.4 fold to 10,000 fold greater HHDH activity as compared to wild type HHDH having the amino acid sequence of SEQ ID NO: 2.

### Claim 4

An isolated or recombinant polypeptide having at least 1.4 fold greater to 10,000 fold greater HHDH activity as compared to wild-type HHDH having the amino acid sequence of SEQ ID NO: 2, and
wherein the polypeptide is encoded by a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleic acid having a sequence selected from the group consisting of SEQ ID NO: 3, 9, 11, 13, 15, 17, 33, 37, 43, 47, 49, 51, 65, 67, 79, 83, 109, 113, 115, 117, 153, 157, 161, 163, 165, 169, 179, 161, 199, 261, 263, 265, 269, 421, 439, 441, 447, 469, 475, 519, 701, 725, 729, 731, 733, 735, 737, and complementary sequences thereof.

### Claim 5 (depends on 1)

An isolated or recombinant polynucleotide encoding the polypeptide of claim 1.

### Claim 6 (depends on 5)

The isolated or recombinant polynucleotide of claim 5, wherein the polynucleotide comprises one or more codons selected from the group consisting of ATT encoding Isoleucine at amino acid position 5 in an encoded HHDH polypeptide; AAG encoding Lysine at amino acid position 36 in an encoded HHDH polypeptide; ATT encoding Isoleucine at amino acid position 63 in an encoded HHDH polypeptide; GAG encoding Glutamic acid at amino acid position 95 in an encoded HHDH polypeptide; and CCC encoding Proline at amino acid position 188 in an encoded HHDH polypeptide;
wherein the amino acid position is the corresponding position in the encoded polypeptide with reference to SEQ ID NO: 2.

### Claim 7 (depends on 5)

An expression vector comprising the polynucleotide of claim 5 operably linked to a promoter.

### Claim 8 (depends on 5)

A host cell transformed with the polynucleotide of claim 5.

### Claim 9 (depends on 8)

A method of making an HHDH polypeptide, said method comprising (a) cultivating the host cell of claim 8 under conditions suitable for production of the HHDH polypeptide, and (b) recovering the HHDH polypeptide.

### Claim 10 (depends on 1)

The isolated polypeptide of claim 1 further having resistance to inhibition by ECAA and the polypeptide, when aligned with SEQ ID NO: 2, having one or more of the residue changes selected from the group consisting of A4V, F82Y, T134V, F136W, F136V, L142R, L178V, W238L, A240T, W249Y and M252I.

### Claim 11 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 4, 12, 16, 18, 34, 38, 44, 48, 52, 66, 80, 84, 114, 154, 158, 170, or 270.

### Claim 12 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 10, 14, or 68, 118, 164, 166, or 180.

### Claim 13 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, when optimally aligned with SEQ ID NO: 2, and which comprises at least one amino acid residue selected from the group consisting of T at (residue) position 2, A or P or S at position 3, V at position 4, D at position 6, either I or F at position 9, L at position 10, S at position 13, S at position 14, K at position 15, C at position 16, T or R at position 17, either C or S or K at position 20, T at position 24, Q at position 26, F at position 28, T at position 29, A at position 30, L at position 31, G at position 33, R at position 34, L at position 35, N at position 36, H at position 37, D at position 40, L at position 44, P at position 45, either P or A at position 47, N at position 52, V at position 54, R at position 55, D at position 56, K at position 58, G or D at position 61, V at position 63, R at position 72, I at position 75, P at position 76, C at position 78, Y at position 82, either S or L at position 84, A at position 85, E at position 91, D at position 93, Q or G at position 95, N at position 96, K at position 107, A at position 112, either T, S or G at position 114, A at position 115, P at position 117, N at position 120, E at position 121, P at position 122, R at position 126, V at position 130, S at position 133, A or V at position 134, L, W or Vat position 136, H at position 139, I or R at position 142, S at position 144, S at position 146, T at position 152, S at position 153, either S or A at position 154, V at position 168, T at position 169, F at position 177, V at position 178, I at position 180, G or I at position 181, K at position 184, Y at position 186, L at position 194, N at position 198, M at position 199, E at position 215, G at position 236, V at position 237, L at position 238, T at position 240, either I or A or V at position 245, Y at position 249, V or I at position 252, and V at position 254.