Variant LOVD polypeptides and their uses
The present disclosure provides acyltransferases useful for synthesizing therapeutically important statin compound.
1. An isolated or recombinant polynucleotide encoding a variant LovD polypeptide having acyltransferase activity, which comprises the amino acid sequence of SEQ ID NO:2 with the mutations L174F and A178L and from 1 to 30 additional mutations, wherein said mutations include K26E.
2. The polynucleotide of claim 1 , wherein the encoded LovD polypeptide amino acid sequence includes the following additional mutations: A123P, N191S/G, A247S and L361M.
3. The polynucleotide of claim 1 , wherein the 1 to 30 additional mutations of the encoded LovD polypeptide amino acid sequence are selected from the group consisting of 14N, A9V, K26E, R28K, R28S, I35L, C40A, C40V, C40F, C40R, S41R, N43R, N43Y, C60F, C60R, C60Y, C60N, C60H, D96R, S109C, A123P, S142N, A184T, A184V, N191S/G, Q241M, A247S, D254E, A261H, A261T, A261E, A261V, L292R, Q295R, Q297E, L335M, L361M, A377V, A383V, N391D, H404K, H404R, Q412R.
4. The polynucleotide of claim 1 , wherein the codons encoding the variant LovD polypeptide sequence have been optimized for expression in a host cell.
5. The polynucleotide of claim 1 , wherein the host cell is E. coli.
6. An expression vector comprising the polynucleotide of claim 1 operably linked to a control sequence suitable for directing expression of the variant LovD polypeptide in a host cell.
7. The expression vector of claim 6 , wherein the control sequence comprises a promoter.
8. An isolated host cell comprising a polynucleotide according to claim 1 .
9. The isolated host cell of claim 8 , wherein the host cell is E. coli.
10. A method of making a variant LovD polypeptide comprising culturing an isolated host cell according to claim 8 under conditions in which the variant LovD polypeptide is expressed.
11. The method of claim 10 which further comprises recovering the variant LovD polypeptide.
12. The method of claim 10 , wherein the isolated host cell is E. coli.
13. An isolated host cell comprising an expression vector of claim 6 .
14. The isolated host cell of claim 13 , wherein the isolated host cell is E. coli.
15. A method of making a variant LovD polypeptide comprising culturing a host cell according to claim 13 under conditions in which the variant LovD polypeptide is expressed.
16. The method of claim 15 which further comprises recovering the variant LovD polypeptide.
17. The method of claim 15 , wherein the host cell is E. coli.