Dehalogenases, nucleic acids encoding them and methods for making and using them
View Patent ↗The invention relates to haloalkane dehalogenases and to polynucleotides encoding the haloalkane dehalogenases. In addition methods of designing new dehalogenases and method of use thereof are also provided. The dehalogenases have increased activity and stability at increased pH and temperature.
1. A synthetic or recombinant nucleic acid operably linked to a promoter, said nucleic acid sequence comprising:
(a) a nucleic acid sequence having at least 97% sequence identity to at least one of SEQ ID NOS: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 45, or 47; or
(b) a nucleic acid sequence complementary to (a); or
(c) the nucleic acid sequence of (a), wherein the nucleic acid encodes at least one polypeptide having a dehalogenase activity.
2. The nucleic acid of claim 1 , wherein the nucleic acid encodes at least one polypeptide having a dehalogenase activity comprising an amino acid sequence having at least 97% sequence identity to at least one of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 46, or 48.
3. The nucleic acid of claim 1 , wherein the nucleic acid sequence has at least 98% sequence identity to at least one of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 45, or 47.
4. The nucleic acid of claim 1 , wherein the nucleic acid sequence has at least 99% sequence identity to at least one of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 45, or 47.
5. The nucleic acid of claim 1 , wherein the nucleic acid sequence comprises a sequence as set forth in at least one of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 45, or 47.
6. The nucleic acid of claim 1 , wherein the dehalogenase activity comprises catalyzing the conversion of a dihalopropionic acid to an optically active halolactic acid.
7. The nucleic acid of claim 1 , wherein optionally the promoter comprises a eukaryotic promoter, a fungal promoter, a mammalian promoter, a prokaryotic promoter or a viral promoter.
8. The nucleic acid of claim 1 , wherein the nucleic acid further comprises a vector.
9. The nucleic acid of claim 8 , wherein the vector is a cloning vector or an expression vector.
10. The nucleic acid of claim 9 , wherein the expression vector is a mammalian expression vector.
11. The nucleic acid of claim 8 , wherein the vector is selected from the group consisting of a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid, a fosmid, a bacteriophage, an artificial chromosome, an adenovirus vector, a retroviral vector, and an adeno-associated viral vector.
12. A host cell comprising a nucleic acid as set forth in claim 1 .
13. The host cell of claim 12 , further comprising a vector.
14. The host cell of claim 12 , wherein the host cell is a prokaryote cell, a eukaryote cell, a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, a plant cell or an insect cell.
15. The nucleic acid of claim 1 wherein the nucleic acid is bound to a solid matrix or a solid phase.
16. The nucleic acid of claim 15 , wherein the solid phase comprises a glass, a polymeric surface, a packed column of polymeric beads, or a magnetic or a paramagnetic particle.
17. The nucleic acid of claim 1 further comprising an antigen or hapten, an antibody, a specific binding fragment, a biotin, an iminobiotin, an avidin, a streptavidin, a sugar, a lectin, an enzyme, an apoenzyme, a cofactor, a complementary homopolymeric oligonucleotide, a hormone, a receptor.
18. A method for generating a dehalogenase-encoding nucleic acid comprising:
(a) providing a parent nucleic acid encoding a dehalogenase comprising a sequence as set forth in claim 1 ; and
(b) modifying the sequence of the parent nucleic acid by a process comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM) or any combination, permutation or iterative process thereof,
thereby generating a dehalogenase-encoding nucleic acid.
19. A method for producing a dehalogenase comprising:
(a) providing a nucleic acid encoding a dehalogenase comprising a sequence as set forth in claim 1 ; and
(b) expressing the nucleic acid to produce a dehalogenase, wherein optionally the nucleic acid is expressed in a host cell.
20. A method for producing a halolactic acid comprising:
(a) providing a nucleic acid encoding a dehalogenase comprising a sequence as set forth in claim 1 , and a dihalopropionic acid;
(b) expressing the nucleic acid to produce a dehalogenase; and
(c) contacting the dihalopropionic acid with the dehalogenase under conditions to produce a halolactic acid, wherein optionally the halolactic acid is an optically active halolactic acid.