Iterative Saturation Mutagenesis Accelerates Laboratory Evolution of Enzyme Stereoselectivity: Rigorous Comparison with Traditional Methods

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• 作者：Manfred T. Reetz ; Shreenath Prasad ; Jos D. Carballeira ; Yosephine Gumulya ; Marco Bocola
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：July 7, 2010
• 年：2010
• 卷：132
• 期：26
• 页码：9144-9152
• 全文大小：294K
• 年卷期：v.132,no.26(July 7, 2010)
• ISSN：1520-5126

文摘

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 10000 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.