Variant LovD polypeptides and their uses
The present disclosure provides acyltransferases useful for synthesizing therapeutically important statin compound.
1. A method of making simvastatin comprising contacting monacolin J or lovastatin substrate with an isolated recombinant variant LovD polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the amino acid sequence of said LovD polypeptide comprises the substitution S172N, in the presence of an α-dimethylbutyryl thioester co-substrate and under conditions in which simvastatin is produced.
2. The method of claim 1 , wherein the monacolin J is a sodium salt.
3. The method of claim 2 , wherein the monacolin J is a sodium salt and activated charcoal is added.
4. The method of claim 1 , wherein the monacolin J is an ammonium salt.
5. The method of claim 4 , wherein an agent for precipitating simvastatin is added.
6. The method of claim 5 , wherein said agent is ammonium hydroxide.
7. The method of claim 1 , wherein said lovastatin substrate, said variant LovD polypeptide and said thioester are added at substantially the same time into a vessel.
8. The method of claim 7 , wherein said lovastatin substrate and said variant LovD polypeptide are first added into a vessel and then said thioester is added into said vessel.
9. The method of claim 1 , wherein an agent for precipitating simvastatin is added.
10. The method of claim 9 , wherein said agent is ammonium hydroxide.