title: Improving catalytic function by ProSAR-driven enzyme evolution.
abstract: We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data. The method thus enables identification of beneficial mutations even in variants with reduced function. We use this hybrid approach to evolve a bacterial halohydrin dehalogenase that improves the volumetric productivity of a cyanation process approximately 4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterol-lowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations.
authors.affiliation: Codexis, Inc., 200 Penobscot Drive, Redwood City, California 94063, USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Fox
authors.forename: S Christopher
authors.initials: SC
authors.lastname: Davis
authors.forename: Emily C
authors.initials: EC
authors.lastname: Mundorff
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Newman
authors.forename: Vesna
authors.initials: V
authors.lastname: Gavrilovic
authors.forename: Steven K
authors.initials: SK
authors.lastname: Ma
authors.forename: Loleta M
authors.initials: LM
authors.lastname: Chung
authors.forename: Charlene
authors.initials: C
authors.lastname: Ching
authors.forename: Sarena
authors.initials: S
authors.lastname: Tam
authors.forename: Sheela
authors.initials: S
authors.lastname: Muley
authors.forename: John
authors.initials: J
authors.lastname: Grate
authors.forename: John
authors.initials: J
authors.lastname: Gruber
authors.forename: John C
authors.initials: JC
authors.lastname: Whitman
authors.forename: Roger A
authors.initials: RA
authors.lastname: Sheldon
authors.forename: Gjalt W
authors.initials: GW
authors.lastname: Huisman
mesh_terms: D000465:Algorithms; D000924:Anticholesteremic Agents / Q000138:chemical synthesis; D000069059:Atorvastatin; D001419:Bacteria / Q000201:enzymology; D002384:Catalysis; D019020:Directed Molecular Evolution / Q000379:methods*; D006538:Heptanoic Acids / Q000138:chemical synthesis; D006867:Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011758:Pyrroles / Q000138:chemical synthesis; D021281:Quantitative Structure-Activity Relationship
chemical_list: D000924:Anticholesteremic Agents; D006538:Heptanoic Acids; D011506:Proteins; D011758:Pyrroles; D000069059:Atorvastatin; D006867:Hydrolases; C430848:halohydrin dehalogenase