Variant LovD polypeptides and their uses
The present disclosure provides acyltransferases useful for synthesizing therapeutically important statin compound.
1. An isolated recombinant variant LovD polypeptide having at least two-fold greater acyltransferase activity than the wild-type Aspergillus terreus acyltransferase, wherein said variant LovD polypeptide is at least 90% sequence identity to SEQ ID NO:2, wherein said variant LovD polypeptide comprises the substitution L174F, wherein the positions is numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2.
2. The variant LovD polypeptide of claim 1 , wherein said polypeptide has at least 10-fold greater acyltransferase activity than the wild-type A. terreus acyltransferase of SEQ ID NO:2.
3. The variant LovD polypeptide of claim 1 , wherein said variant further comprises 1 to 30 additional mutations.
4. The variant LovD polypeptide of claim 3 , wherein said 1 to 30 further additional mutations are selected from C40A, C40V, C40F, C40R, S41R, N43Y, C60F, C60R, C60Y, C60N, C60H, S142N, A184T, A184V, D254E, A261T, A261E, A261V, L292R, Q295R, A377V, and Q412R.
5. A method of making simvastatin comprising contacting monacolin J substrate with the variant LovD polypeptide of claim 1 , in the presence of an α-dimethylbutyryl thioester co-substrate and under conditions in which the monacolin J is converted to simvastatin.
6. The method of claim 5 , wherein the monacolin J is a sodium salt.
7. The method of claim 6 , wherein the monacolin J is a sodium salt and activated charcoal is added.
8. The method of claim 5 , wherein the monacolin J is an ammonium salt.
9. The method of claim 5 , wherein an agent for precipitating simvastatin is added.
10. The method of claim 9 , wherein said agent is ammonium hydroxide.
11. A method of making simvastatin comprising contacting a lovastatin substrate with the variant LovD polypeptide of claim 1 , in the presence of an α-dimethylbutyryl thioester co-substrate and under conditions in which the lovastatin substrate is converted to simvastatin.
12. The method of claim 11 , wherein said lovastatin substrate, said variant LovD polypeptide and said thioester are charges at substantially the same time into a vessel.
13. The method of claim 11 , wherein said lovastatin substrate and said variant LovD polypeptide are first charged into a vessel and then said thioester is charged into said vessel.
14. The method of claim 11 , wherein an agent for precipitating simvastatin is added.
15. The method of claim 14 , wherein said agent is ammonium hydroxide.