Variant LovD polypeptide
View Patent ↗The invention disclosed herein relates to methods and materials for producing simvastatin and related compounds such as huvastatin. In particular, the disclosure teaches that variants of the LovD acyltransferase polypeptide can be engineered to exhibit properties that facilitate their use in the production of simvastatin and/or huvastatin. The materials and processes disclosed herein are designed so that fermentation facilities currently producing lovastatin can be converted to producing simvastatin and related compounds with minimal modifications.
1. An isolated variant of a native LovD polypeptide, wherein the variant LovD polypeptide comprises amino acid substitutions at an amino acid residue selected from the group consisting of: A10; D12; K26; A86; A190; H161; K227; G275; V334; and L361 of SEQ ID NO: 1, wherein the variant LovD polypeptide has an improved whole cell activity at 25° C. as compared to the LovD polypeptide of SEQ ID NO:1.
2. The variant LovD polypeptide of claim 1 , wherein
the variant comprises amino substitutions at amino acid residue positions C40 and C60 of SEQ ID NO:1.
3. The variant LovD polypeptide of claim 1 or 2 , wherein the variant LovD polypeptide comprises amino acid substitutions selected from the group consisting of: A10V; D12G; K26E; C40A; C60N; A86V; H161Y; A190T; K227R; G275S; V334D; V334F; and L361M.
4. The variant LovD polypeptide of claim 1 , wherein the variant exhibits:
a decreased aggregation;
an improved thermal stability;
an improved catalytic activity;
an improved k cat /K m value; or
an improved soluble expression level.
5. A composition of matter comprising:
monacolin J;
an acyl thioester that donates an acyl moiety to the C8 hydroxyl group of monacolin J in the presence LovD acyltransferase;
a variant of LovD polypeptide comprising an amino acid substitution at:
amino acid residue positions C40 and C60; and
at least one amino acid residue position: A10; D12; K26; A86; A190; H161; K227; G275; V334; or L361 of SEQ ID NO:1, wherein the variant LovD polypeptide has an improved whole cell activity at 25° C. as compared to the LovD polypeptide of SEQ ID NO:1; and
simvastatin.