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Less Unfavorable Salt Bridges on the Enzyme Surface Result in More Organic Cosolvent Resistance
2021, Cui, Haiyang, Eltoukhy, Lobna, Zhang, Lingling, Markel, Ulrich, Jaeger, Karl-Erich, Davari, Mehdi D., Schwaneberg, Ulrich
Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems. Iterative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance toward three OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and half-life up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50–100 °C. The salt bridge redesign provides protein engineers with a powerful and likely general approach to design OSs- and/or thermal-resistant lipases and other α/β-hydrolases. © 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
A Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT
2021, Markel, Ulrich, Lanvers, Pia, Sauer, Daniel F., Wittwer, Malte, Dhoke, Gaurao V., Davari, Mehdi D., Schiffels, Johannes, Schwaneberg, Ulrich
Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases. © 2020 The Authors. Published by Wiley-VCH GmbH
How to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study
2020, Cui, Haiyang, Stadtmüller, Tom H.J., Jiang, Qianjia, Jaeger, Karl-Erich, Schwaneberg, Ulrich, Davari, Mehdi D.
Expanding synthetic capabilities to routinely employ enzymes in organic solvents (OSs) is a dream for protein engineers and synthetic chemists. Despite significant advances in the field of protein engineering, general and transferable design principles to improve the OS resistance of enzymes are poorly understood. Herein, we report a combined computational and directed evolution study of Bacillus subtlis lipase A (BSLA) in three OSs (i. e., 1,4-dioxane, dimethyl sulfoxide, 2,2,2-trifluoroethanol) to devise a rational strategy to guide engineering OS resistant enzymes. Molecular dynamics simulations showed that OSs reduce BSLA activity and resistance in OSs by (i) stripping off essential water molecules from the BLSA surface mainly through H-bonds binding; and (ii) penetrating the substrate binding cleft leading to inhibition and conformational change. Interestingly, integration of computational results with “BSLA-SSM” variant library (3439 variants; all natural diversity with amino acid exchange) revealed two complementary rational design strategies: (i) surface charge engineering, and (ii) substrate binding cleft engineering. These strategies are most likely applicable to stabilize other lipases and enzymes and assist experimentalists to design organic solvent resistant enzymes with reduced time and screening effort in lab experiments. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA
Computer-Assisted Recombination (CompassR) Teaches us How to Recombine Beneficial Substitutions from Directed Evolution Campaigns
2020, Cui, Haiyang, Cao, Hao, Cai, Haiying, Jaeger, Karl-Erich, Davari, Mehdi D., Schwaneberg, Ulrich
A main remaining challenge in protein engineering is how to recombine beneficial substitutions. Systematic recombination studies show that poorly performing variants are usually obtained after recombination of 3 to 4 beneficial substitutions. This limits researchers in exploiting nature's potential in generating better enzymes. The Computer-assisted Recombination (CompassR) strategy provides a selection guide for beneficial substitutions that can be recombined to gradually improve enzyme performance by analysis of the relative free energy of folding (ΔΔGfold). The performance of CompassR was evaluated by analysis of 84 recombinants located on 13 positions of Bacillus subtilis lipase A. The finally obtained variant F17S/V54K/D64N/D91E had a 2.7-fold improved specific activity in 18.3 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). In essence, the deducted CompassR rule allows recombination of beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time-efficient manner. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.