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

Abstract

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.

Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance

“…To achieve this, simultaneous saturation mutagenesis (SSM) 17,18) was applied. A synergistic effect on enzymatic activity would be expected following multiple mutations in the active site, 19,20) but this is difficult to obtain via the iterative site-directed saturation mutagenesis performed in previous studies. 14,16) …”

Directed Divergent Evolution of a Thermostable D‐Tagatose Epimerase towards Improved Activity for Two Hexose Substrates

“…Directed evolution, relying on iterative cycles of mutagenesis and screening of the resulting libraries for variants that exhibit the desired traits [2] , has been shown to allow improving almost any property that is of importance for an industrially useful biocatalyst, including thermostability [3] , enantioselectivity [4] or catalytic rate [5] .…”

“…This enzyme shows promiscuous enzymatic activity for C3 epimerization of all 4 therefore an interesting starting point for further catalytic rate optimization. We decided to divergently evolve this thermostable Var8 for improved production of two rare ketohexoses, specifically D-psicose from D-fructose and L-tagatose from L-sorbose.…”

Laboratory‐Evolved Enzymes Provide Snapshots of the Development of Enantioconvergence in Enzyme‐Catalyzed Epoxide Hydrolysis

“…We have obtained, by ISM‐driven5 directed evolution, enzyme variants that were able to catalyze the formation of the hydrolysis product ( 2 ) with enriched optical purity 2, 3. We also isolated laboratory‐evolved variants that catalyze the production of enriched ( R )‐ 2 from racemic 1 .…”