Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?
Congratulations to Ulrich Markel and Leilei Zhu on their recent publication!
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 compare 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 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, 2,2,2-trifluoroethanol, and dimethyl sulfoxide. Our analysis revealed that although a significant amount of all possible single substitutions contributes to improved cosolvent resistance, only a fraction of these substitutions could be detected in the three random mutagenesis libraries. 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, our investigation of the BSLA SSM library indicates the importance of introducing surface charges for organic cosolvent resistance.
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Markel, U., Zhu, L., Frauenkron-Machedjou, V. J., Zhao, J., Bocola, M., Davari, M. D., Jaeger, K.-E., Schwaneberg, U. (2017). Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents? Catalysts, 7, 142. DOI:10.3390/catal7050142