Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?
Ulrich Markel,Leilei Zhu,Victorine Josiane Frauenkron-Machedjou,Jing Zhao,Marco Bocola,Mehdi D. Davari,Karl-Erich Jaeger,Ulrich Schwaneberg +7 more
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TL;DR: This is the first study that quantifies the capability of these diversity generation methods generally employed in directed evolution campaigns and compares them to the entire natural diversity with a single substitution.
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Abstract: Despite the significant advances in the field of protein engineering, general design principles to improve organic cosolvent resistance of enzymes still remain undiscovered. Previous studies drew conclusions to engineer enzymes for their use in water-miscible organic solvents based on few amino acid substitutions. In this study, we conduct a comparison of a Bacillus subtilis lipase A (BSLA) library—covering the full natural diversity of single amino acid substitutions at all 181 positions of BSLA—with three state of the art random mutagenesis methods: error-prone PCR (epPCR) with low and high mutagenesis frequency (epPCR-low and high) as well as a transversion-enriched Sequence Saturation Mutagenesis (SeSaM-Tv P/P) method. Libraries were searched for amino acid substitutions that increase the enzyme’s resistance to the water-miscible organic cosolvents 1,4-dioxane (DOX), 2,2,2-trifluoroethanol (TFE), and dimethyl sulfoxide (DMSO). Our analysis revealed that 5%–11% of all possible single substitutions (BSLA site-saturation mutagenesis (SSM) library) contribute to improved cosolvent resistance. However, only a fraction of these substitutions (7%–12%) could be detected in the three random mutagenesis libraries. To our knowledge, this is the first study that quantifies the capability of these diversity generation methods generally employed in directed evolution campaigns and compares them to the entire natural diversity with a single substitution. Additionally, the investigation of the BSLA SSM library revealed only few common beneficial substitutions for all three cosolvents as well as the importance of introducing surface charges for organic cosolvent resistance—most likely due to a stronger attraction of water molecules.
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Machine learning-assisted enzyme engineering.
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Advances in Recombinant Lipases : Production, Engineering, Immobilization and Application in the Pharmaceutical Industry
Fabiano Jares Contesini,Marcelo Gomes Davanço,Gustavo Pagotto Borin,Katherina Garcia Vanegas,João Pedro Gonçalves Cirino,Ricardo Rodrigues de Melo,Uffe Hasbro Mortensen,Kristiina Hildén,Daniel Rossi Campos,Patrícia de Oliveira Carvalho +9 more
TL;DR: Approaches for strain and protein engineering, immobilization and the application of lipases in the pharmaceutical industry are reviewed.
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Enzyme Hydration Determines Resistance in Organic Cosolvents
Haiyang Cui,Lingling Zhang,Lobna Eltoukhy,Qianjia Jiang,Seval Kübra Korkunç,Karl-Erich Jaeger,Karl-Erich Jaeger,Ulrich Schwaneberg,Ulrich Schwaneberg,Mehdi D. Davari +9 more
TL;DR: In this paper, the authors describe the use of biocatalysis in organic solvents (OSs) for organic synthesis and for the production of pharmaceuticals, flavors, and fragrances.
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How to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study
Haiyang Cui,Tom H. J. Stadtmüller,Qianjia Jiang,Karl-Erich Jaeger,Ulrich Schwaneberg,Ulrich Schwaneberg,Mehdi D. Davari +6 more
TL;DR: Interestingly, integration of computational results with “BSLA‐SSM” variant library revealed two complementary rational design strategies: surface charge engineering, and substrate binding cleft engineering, which are most likely applicable to stabilize other lipases and enzymes.
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