Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity: rigorous comparison with traditional methods.
Efficacy in laboratory evolution of enzymes is currently a pressing issue, making comparative studies of different methods and strategies mandatory. Recent reports indicate that iterative saturation mutagenesis (ISM) provides a means to accelerate directed evolution of stereoselectivity and thermostability, but statistically meaningful comparisons with other methods have not been documented to date. In the present study, the efficacy of ISM has been rigorously tested by applying it to the previously most systematically studied enzyme in directed evolution, the lipase from Pseudomonas aeruginosa as a catalyst in the stereoselective hydrolytic kinetic resolution of a chiral ester. Upon screening only 10,000 transformants, unprecedented enantioselectivity was achieved (E = 594). ISM proves to be considerably more efficient than all previous systematic efforts utilizing error-prone polymerase chain reaction at different mutation rates, saturation mutagenesis at hot spots, and/or DNA shuffling, pronounced positive epistatic effects being the underlying reason.
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany. reetz@mpi-muelheim.mpg.de
Manfred T
MT
Reetz
Shreenath
S
Prasad
José D
JD
Carballeira
Yosephine
Y
Gumulya
Marco
M
Bocola
D055162:Biocatalysis; D019020:Directed Molecular Evolution / Q000379:methods*; D004798:Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013237:Stereoisomerism; D013379:Substrate Specificity
D004798:Enzymes; D008049:Lipase