Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?

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. © 2017 by the authors

Engineering a lipase for organic cosolvent resistance - How do current directed evolution approaches compete with the potential that nature offers?

Our desire to design enzymes resistant to organic cosolvents is still challenged by our level of molecular understanding of this important issue. This is why currently directed evolution is utilized as the method of choice to discover promising enzyme variants. As directed evolution-based studies typically report only few beneficial amino acid exchanges, the deduction of general principles for the design of enzymes with increased resistance to water/organic solvent mixtures is challenging. Here, we present the comparative analysis of a Bacillus subtilis lipase A (BSLA) library, covering the full diversity of single amino acid exchanges at all 181 positions of BSLA (BSLA SSM library), and three random mutagenesis libraries (error-prone PCR with low and high mutagenesis frequencies, as well as a transversion-enriched Sequence Saturation Mutagenesis (SeSaM-Tv P/P) library). Screening of the BSLA SSM library for resistance to the water-miscible organic cosolvents 1,4‑dioxane (DOX), 2,2,2 trifluoroethanol (TFE), and dimethyl sulfoxide (DMSO) revealed that 5 – 11% of all possible single substitutions promote organic cosolvent resistance. However, only 7 – 12% of these beneficial substitutions were identified in the three random mutagenesis libraries. To our knowledge, this is the first study quantifying the number of beneficial substitutions obtainable by random mutagenesis compared to the total number of beneficial single-substitutions (BSLA SSM library). Moreover, comprehensive analysis of the BSLA SSM library revealed that only few beneficial amino acid substitutions were common for all three organic cosolvents tested. These findings illustrate that – even when the total single-substitution diversity is available – our understanding of organic cosolvent resistance still remains incomplete. Hence, deducing general design principles based on relatively few amino acid exchanges, as it is common practice in directed evolution campaigns, seems counterintuitive. Furthermore, analysis of the BSLA SSM library conferred valuable insights into the role of surface-exposed charges for organic cosolvent resistance. Structural inspection of beneficial variants revealed that this is due to the attraction of water rather than to the formation of salt bridges. Please click Additional Files below to see the full abstract

Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole Amidation

Here we report a cytochrome P450 variant that catalyzes C_2-amidation of 1-methylindoles with tosyl azide via nitrene transfer. Before evolutionary optimization, the enzyme exhibited two undesired side reactivities resulting in reduction of the putative iron-nitrenoid intermediate or cycloaddition between the two substrates to form triazole products. We speculated that triazole formation was a promiscuous cycloaddition activity of the P450 heme domain, while sulfonamide formation likely arose from surplus electron transfer from the reductase domain. Directed evolution involving mutagenesis of both the heme and reductase domains delivered an enzyme providing the desired indole amidation products with up to 8400 turnovers, 90% yield, and a shift in chemoselectivity from 2:19:1 to 110:12:1 in favor of nitrene transfer over reduction or triazole formation. This work expands the substrate scope of hemoprotein nitrene transferases to heterocycles and highlights the adaptability of the P450 scaffold to solve challenging chemoselectivity problems in non-natural enzymatic catalysis

Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole Amidation

Here we report a cytochrome P450 variant that catalyzes C_2-amidation of 1-methylindoles with tosyl azide via nitrene transfer. Before evolutionary optimization, the enzyme exhibited two undesired side reactivities resulting in reduction of the putative iron-nitrenoid intermediate or cycloaddition between the two substrates to form triazole products. We speculated that triazole formation was a promiscuous cycloaddition activity of the P450 heme domain, while sulfonamide formation likely arose from surplus electron transfer from the reductase domain. Directed evolution involving mutagenesis of both the heme and reductase domains delivered an enzyme providing the desired indole amidation products with up to 8400 turnovers, 90% yield, and a shift in chemoselectivity from 2:19:1 to 110:12:1 in favor of nitrene transfer over reduction or triazole formation. This work expands the substrate scope of hemoprotein nitrene transferases to heterocycles and highlights the adaptability of the P450 scaffold to solve challenging chemoselectivity problems in non-natural enzymatic catalysis

An artificial ruthenium-containing β-barrel protein for alkene–alkyne coupling reaction

A modified Cp*Ru complex, equipped with a maleimide group, was covalently attached to a cysteine of an engineered variant of Ferric hydroxamate uptake protein component: A (FhuA). This synthetic metalloprotein catalyzed the intermolecular alkene–alkyne coupling of 3-butenol with 5-hexynenitrile. When compared with the protein-free Cp*Ru catalyst, the biohybrid catalyst produced the linear product with higher regioselectivity