Improving catalytic function by ProSAR-driven enzyme evolution.
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.
Fox
Richard J
RJ
Codexis, Inc., 200 Penobscot Drive, Redwood City, California 94063, USA.
Davis
S Christopher
SC
Mundorff
Emily C
EC
Newman
Lisa M
LM
Gavrilovic
Vesna
V
Ma
Steven K
SK
Chung
Loleta M
LM
Ching
Charlene
C
Tam
Sarena
S
Muley
Sheela
S
Grate
John
J
Gruber
John
J
Whitman
John C
JC
Sheldon
Roger A
RA
Huisman
Gjalt W
GW
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
D000924:Anticholesteremic Agents; D006538:Heptanoic Acids; D011506:Proteins; D011758:Pyrroles; D000069059:Atorvastatin; D006867:Hydrolases; C430848:halohydrin dehalogenase