# US 2019/0169580 A1 · VARIANT LovD POLYPEPTIDE > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0169580 A1 - Document ID: US20190169580A1 - Status: Published application - Kind: A1 - Application number: 16/277,921 - Filed: 2019-02-15 - Published: 2019-06-06 - Earliest claimed: 2009-10-08 - Assignees: The Regents of the University of California - Applicants: The Regents of the University of California - Inventors: Yi Tang; Xue Gao; Xinkai Xie - Web page: https://patentgrep.com/patent/US20190169580A1 ## CPC - C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)} - C07K 2319/00: Fusion polypeptide - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract 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 lovastatin being converted to simvastatin and related compounds with minimal modifications. ## Claims ### Claim 1 A method of making a variant of a LovD polypeptide shown in SEQ ID NO:1 comprising: using a polynucleotide mutagenesis procedure to generate a population of mutants of the LovD polynucleotide shown in SEQ ID NO: 5, wherein the population of mutant LovD polynucleotides encodes LovD polypeptide variants having at least one amino acid substitution selected from the group consisting of K26E, G275S, or L361M; and expressing a population of LovD polypeptide variants encoded by the population of LovD polynucleotide mutants; so that a variant of a LovD polypeptide shown in SEQ ID NO:1 is made. ### Claim 2 (depends on 1) The method of claim 1, wherein the polynucleotide mutagenesis procedure is controlled so as to generate a substitution mutation at a codon that encodes an amino acid residue selected from the group consisting of: A10; D12; A86; A190; H161; K227; and V334 of SEQ ID NO:1. ### Claim 3 (depends on 1) The method of claim 1, wherein the polynucleotide mutagenesis procedure is controlled so as to generate a variant comprising C40A and C60N; and a further a substitution mutation at a codon that encodes 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. ### Claim 4 (depends on 1) The method of claim 1, wherein the polynucleotide mutagenesis procedure comprises a saturation mutagenesis or an error prone polymerase chain reaction procedure. ### Claim 5 (depends on 1) The method of claim 1, further comprising screening one or more members of the population of LovD polypeptide variants so as to identify a variant that exhibits: a decreased aggregation; an improved thermal stability; an improved acyltransferase activity; an improved kcat/Krn value; an improved soluble expression level; and/or an improved whole cell activity at 25° C., as compared to the LovD polypeptide shown in SEQ ID NO:1. ### Claim 6 A polynucleotide encoding a variant LovD polypeptide, wherein: the variant LovD polypeptide comprises a functional LovD acyltransferase with an amino acid sequence having at least 90% identity to SEQ ID NO:1; and the variant LovD polypeptide comprises an amino acid substitution at amino acid residue position selected from the group consisting of: A10; D12; K26; A86; A190; H161; K227; G275; V334; and L361 of SEQ ID NO: 1. ### Claim 7 (depends on 6) The polynucleotide of claim 6, wherein the variant LovD polypeptide comprises an amino acid substitution selected from the group consisting of: A10V; D12G; K26E; A86V; H161Y; A190T; K227R; G275S; V334D; V334F; and L361M of SEQ ID NO:1. ### Claim 8 (depends on 6) The polynucleotide of claim 6, wherein the variant LovD polypeptide comprises at least two amino acid substitutions at amino acid residue position selected from the group consisting of: A10; D12; K26; A86; A190; H161; K227; G275; V334; and L361 of SEQ ID NO: 1. ### Claim 9 (depends on 6) The polynucleotide of claim 6, 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 of SEQ ID NO:1. ### Claim 10 (depends on 8) The polynucleotide of claim 8, wherein the at least two amino acid substitutions of the variant LovD polypeptide include at least one amino acid residue position selected from the group consisting of: K26, G275, and L361 of SEQ ID NO:1. ### Claim 11 (depends on 8) The polynucleotide of claim 8, wherein the at least two amino acid substitutions of the variant LovD polypeptide include at least one amino acid residue selected from the group consisting of: K26E, G275S, and L361M of SEQ ID NO:1. ### Claim 12 (depends on 6) The polynucleotide of claim 6, wherein the variant LovD polypeptide comprises amino acid substitution at amino acid residue position D12 of SEQ ID NO:1. ### Claim 13 (depends on 12) The polynucleotide of claim 12, wherein the variant LovD polypeptide further comprises an amino acid substitution selected from the group consisting of: K26, G275, and L361 of SEQ ID NO:1. ### Claim 14 (depends on 6) The polynucleotide of claim 6, wherein the variant LovD polypeptide further comprises an amino substitution at amino acid residue positions C40 and/or C60 of SEQ ID NO:1. ### Claim 15 (depends on 6) The polynucleotide of claim 6, wherein the variant LovD polypeptide exhibits: a decreased aggregation; an improved catalytic activity; an improved thermal stability; an improved kat/Km value; an improved soluble expression level; or an improved whole cell activity at 25° C.; as compared to the LovD polypeptide shown in SEQ ID NO:1. ### Claim 16 (depends on 6) A vector comprising the polynucleotide of claim 6. ### Claim 17 (depends on 16) The vector of claim 16 operably linked to control sequences recognized by a host cell transformed with the vector. ### Claim 18 (depends on 17) A host cell comprising the vector of claim 17. ### Claim 19 (depends on 18) The host cell of claim 18 wherein the host cell is selected from the group consisting of an Escherichia coli, Apergillus terreus, Monascus ruber, Monascus puipureus, Monascus pilosus, Monascus vitreus, Monascus pubigerus, Candida cariosilognicola, Aspergillus oryzea, Doratomyces stemonitis, Paecilomyces virioti, Penicillum citrinum, Penicillin chysogenum, Scopulariopsis brevicaulis, and Trichoderma viride. ### Claim 20 (depends on 6, 19) A process for producing a variant LovD polypeptide comprising culturing the host cell of claim 19 under conditions suitable for expression of a polypeptide encoded by a polynucleotide of claim 6 so that the variant LovD polypeptide is produced.