Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?
AbstractDespite 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. View Full-Text
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Markel, U.; Zhu, L.; Frauenkron-Machedjou, V.J.; Zhao, J.; Bocola, M.; Davari, M.D.; Jaeger, K.-E.; Schwaneberg, U. Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents? Catalysts 2017, 7, 142.
Markel U, Zhu L, Frauenkron-Machedjou VJ, Zhao J, Bocola M, Davari MD, Jaeger K-E, Schwaneberg U. Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents? Catalysts. 2017; 7(5):142.Chicago/Turabian Style
Markel, Ulrich; Zhu, Leilei; Frauenkron-Machedjou, Victorine J.; Zhao, Jing; Bocola, Marco; Davari, Mehdi D.; Jaeger, Karl-Erich; Schwaneberg, Ulrich. 2017. "Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?" Catalysts 7, no. 5: 142.
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