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

Deciphering rationales that determine the resistance of a lipase towards non-conventional media

The naturally existing enzymes have been evolved to work efficiently in aqueous environment of their hosts. Very often, their activity gets dramatically lost in the presence of non-conventional media (e.g. ionic liquids (ILs) and organic solvents (OS)) which are often present in industrial processes. Therefore, it is crucial to maintain/improve the enzyme resistance towards non-conventional media for the efficient operation of enzymes in industrial process conditions. Despite of the many successful stories of improving enzyme resistance towards many non-conventional media by protein engineering, there have been no geneneral principles for guiding researchers for efficient reengineering of enzymes. The aim of this work was to obtain protein engineering principles for improving IL and OS resistance of enzymes. A systematic study including site saturation mutagenesis (SSM) library generation of the complete sequence of a model enyzme Bacillus subtilis LipA (BSLA) and screening in presence of four ILs ([BMIM][Cl], [BMIM][Br], [BMIM][I], [BMIM][TfO]) and three OS (DMSO, TFE, 1,4-dioxane) were performed. The generation of 181 SSM-BSLA libraries was carried out by two PhD fellows in close cooperation (91 SSM-BSLA libraries by Victorine Josiane Frauenkron Machedjou and 90 by Alexander Fulton). Overall, 18547 clones were generated and screened which contained all possible 3620 single mutations as confirmed by sequencing. The number of beneficial positions and substitutions, type of beneficial amino acid exchange and location of positions was investigated. To the best of our knowledge, this is the first study based on such a complete set of experimental data to investigate IL/OS resistance of an enzyme.In a first study (described in chapter 3), the resistance of SSM-BSLA variants towards four [BMIM]-based ILs (Cl-, Br-, I-, and TfO-) was investigated. The highest number of improved variants was obtained for [BMIM][Cl]: 69% of all positions and 13% of all substitutions showed improvements. For the four ILs, at 50-69% of all positions at least one substitution led to improvement whereas only 6 13% of all substitutions displayed increased resistance in all four ILs. It was also noteworthy that higher improvements from single substitution (>2.5-fold) were difficult to achieve by screening random mutagenesis library since only 0.3% of all the substitutions resulted in improvement >2.5-fold. Furthermore, amino acid substitutions with polar and charged amino acids were demonstrated to considerably enhance ILs resistance of BSLA. Among the best variants, the highest improvement (11.3-fold) was obtained for [BMIM][Cl] for a substitution from charged acidic to aromatic (Asp91Trp). Two main lessons were obtained: a) resistance improvements were obtainable at 50-69% of all amino acid positions, explaining the success rate of small sized random mutant libraries; b) in total 6-13% of substitutions led to improved resistance. Among the beneficial substitutions 66-95% were substituted by chemically different amino acids (e.g. aromatic to polar/aliphatic/charged amino acids), indicating that mutagenesis methods introducing transversions should at least for lipases like BSLA be favored to improve IL resistance.In a second study (described in chapter 4), the resistance pattern of SSM-BSLA variants towards three OS (DMSO, TFE, and 1,4-dioxane) was investigated. For 1,4-dioxane and TFE, 4-5% substitutions at 41% positions led to improved resistance. As observed for IL resistance of BSLA, improvements >2.5-fold were difficult to achieve with single point mutation, only 0.03% and 0.17% variants were obtained for DMSO / 1,4-dioxane and TFE, respectively. In addition, substitutions of BSLA WT amino acid residues with charged amino acids residues predominantly led to improved OS resistance towards TFE and 1,4-dioxane, whereas polar substitutions were preferred for DMSO.Overall, two features for BSLA resistance in non-conventional media were observed: 1) resistance improvements were obtainable at >41% (in OS) and >50% (in ILs) of all amino acid positions of BSLA, indicating why improved enzyme variants can often be identified from small sized random mutant libraries (1000-2000 clones) and 2) often only few substitutions per amino acid positions led to improved resistance (4-13% of all substitutions) for OS and ILs. Among the beneficial substitutions, 66-95% and 58-95% were exchanged with chemically different amino acids (e.g. aromatic amino acids substituted by polar/aliphatic/charged amino acid) for ILs and OS, respectively. The latter indicates that mutagenesis methods that introduce transversions should, at least for lipases like BSLA be favored to generate variants with improved OS and/or IL resistance. Furthermore, the highest number of improved BSLA variants were obtained for [BMIM][Cl] and DMSO among the four ILs and the three OS, respectively

Deciphering rationales that determine the resistance of a lipase towards non-conventional media

The naturally existing enzymes have been evolved to work efficiently in aqueous environment of their hosts. Very often, their activity gets dramatically lost in the presence of non-conventional media (e.g. ionic liquids (ILs) and organic solvents (OS)) which are often present in industrial processes. Therefore, it is crucial to maintain/improve the enzyme resistance towards non-conventional media for the efficient operation of enzymes in industrial process conditions. Despite of the many successful stories of improving enzyme resistance towards many non-conventional media by protein engineering, there have been no geneneral principles for guiding researchers for efficient reengineering of enzymes. The aim of this work was to obtain protein engineering principles for improving IL and OS resistance of enzymes. A systematic study including site saturation mutagenesis (SSM) library generation of the complete sequence of a model enyzme Bacillus subtilis LipA (BSLA) and screening in presence of four ILs ([BMIM][Cl], [BMIM][Br], [BMIM][I], [BMIM][TfO]) and three OS (DMSO, TFE, 1,4-dioxane) were performed. The generation of 181 SSM-BSLA libraries was carried out by two PhD fellows in close cooperation (91 SSM-BSLA libraries by Victorine Josiane Frauenkron Machedjou and 90 by Alexander Fulton). Overall, 18547 clones were generated and screened which contained all possible 3620 single mutations as confirmed by sequencing. The number of beneficial positions and substitutions, type of beneficial amino acid exchange and location of positions was investigated. To the best of our knowledge, this is the first study based on such a complete set of experimental data to investigate IL/OS resistance of an enzyme.In a first study (described in chapter 3), the resistance of SSM-BSLA variants towards four [BMIM]-based ILs (Cl-, Br-, I-, and TfO-) was investigated. The highest number of improved variants was obtained for [BMIM][Cl]: 69% of all positions and 13% of all substitutions showed improvements. For the four ILs, at 50-69% of all positions at least one substitution led to improvement whereas only 6 13% of all substitutions displayed increased resistance in all four ILs. It was also noteworthy that higher improvements from single substitution (>2.5-fold) were difficult to achieve by screening random mutagenesis library since only 0.3% of all the substitutions resulted in improvement >2.5-fold. Furthermore, amino acid substitutions with polar and charged amino acids were demonstrated to considerably enhance ILs resistance of BSLA. Among the best variants, the highest improvement (11.3-fold) was obtained for [BMIM][Cl] for a substitution from charged acidic to aromatic (Asp91Trp). Two main lessons were obtained: a) resistance improvements were obtainable at 50-69% of all amino acid positions, explaining the success rate of small sized random mutant libraries; b) in total 6-13% of substitutions led to improved resistance. Among the beneficial substitutions 66-95% were substituted by chemically different amino acids (e.g. aromatic to polar/aliphatic/charged amino acids), indicating that mutagenesis methods introducing transversions should at least for lipases like BSLA be favored to improve IL resistance.In a second study (described in chapter 4), the resistance pattern of SSM-BSLA variants towards three OS (DMSO, TFE, and 1,4-dioxane) was investigated. For 1,4-dioxane and TFE, 4-5% substitutions at 41% positions led to improved resistance. As observed for IL resistance of BSLA, improvements >2.5-fold were difficult to achieve with single point mutation, only 0.03% and 0.17% variants were obtained for DMSO / 1,4-dioxane and TFE, respectively. In addition, substitutions of BSLA WT amino acid residues with charged amino acids residues predominantly led to improved OS resistance towards TFE and 1,4-dioxane, whereas polar substitutions were preferred for DMSO.Overall, two features for BSLA resistance in non-conventional media were observed: 1) resistance improvements were obtainable at >41% (in OS) and >50% (in ILs) of all amino acid positions of BSLA, indicating why improved enzyme variants can often be identified from small sized random mutant libraries (1000-2000 clones) and 2) often only few substitutions per amino acid positions led to improved resistance (4-13% of all substitutions) for OS and ILs. Among the beneficial substitutions, 66-95% and 58-95% were exchanged with chemically different amino acids (e.g. aromatic amino acids substituted by polar/aliphatic/charged amino acid) for ILs and OS, respectively. The latter indicates that mutagenesis methods that introduce transversions should, at least for lipases like BSLA be favored to generate variants with improved OS and/or IL resistance. Furthermore, the highest number of improved BSLA variants were obtained for [BMIM][Cl] and DMSO among the four ILs and the three OS, respectively

Exploring the full natural diversity of single amino acid exchange reveals that 40-60% of BSLA positions improve organic solvents resistance

Abstract Objectives Protein engineering has been employed to successfully improve organic solvent resistance of enzymes. Exploration of nature’s full potential (how many beneficial positions/beneficial substitutions of the target enzyme) to improve organic solvent resistance of enzymes by a systematic study was performed. Results We report the results of screening the previously generated BSLA (Bacillus subtilis lipase A)-SSM (site saturation mutagenesis) library (covering the full natural diversity of BSLA with one amino acid exchange) in presence of three cosolvents. The potential of single amino acid substitution that nature offers to improve the cosolvent resistance of BSLA was determined by analyzing the number of beneficial positions/substitutions, accessibility and chemical compositions. Conclusion Lessons learned from analysis of BSLA-SSM library are: (1) 41–59% of BSLA positions with in total 4–10% of all possible substitutions improve the cosolvent resistance against TFE, DOx, and DMSO; (2) charged substitutions are preferred to improve DOx and TFE resistance whereas polar ones are preferred for DMSO; (3) charged substitutions on the surface predominantly improved resistance while polar ones were preferred in buried “regions”. (4) Interestingly, 58–93% of beneficial substitutions led to chemically different amino acids

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

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

Unraveling the effects of amino acid substitutions enhancing lipase resistance to an ionic liquid: a molecular dynamics study

Understanding of the structural and dynamic properties of enzymes in non-aqueous media (e.g., ionic liquids, ILs) is highly attractive for protein engineers and synthetic biochemists. Despite a growing number of molecular dynamics (MD) simulation studies on the influence of different ILs on wild-type enzymes, the effects of various amino acid substitutions on the stability and activity of enzymes in ILs remain to be unraveled at the molecular level. Herein, we selected fifty previously reported Bacillus subtilis lipase A (BSLA) variants with increased resistance towards an IL (15 vol% 1-butyl-3-methylimidazolium trifluoromethanesulfonate; [Bmim][TfO]), and also ten non-resistant BSLA variants for a MD simulation study to identify the underlying molecular principles. Some important properties differentiating resistant and non-resistant BSLA variants from wild-type were elucidated. Results show that, in 15 vol% [Bmim][TfO] aqueous solution, 40% and 60% of non-resistant variants have lower and equal probabilities to form a catalytically important hydrogen bond between S77 and H156 compared to wild-type, whereas 36% and 56% of resistant variants show increased and equal probabilities, respectively. Introducing positively charged amino acids close to the substrate-binding cleft for instance I12R is beneficial for the BSLA resistance towards 15 vol% [Bmim][TfO], likely due to the reduced probability of [Bmim]+ cations clustering near the cleft. In contrast, substitution with a large hydrophobic residue like I12F can block the cleft through hydrophobic interaction with a neighboring nonpolar loop 134–137 or/and an attractive π–π interaction with [Bmim]+ cations. In addition, the resistant variants having polar substitutions on the surface show higher ability to stabilize the surface water molecule network in comparison to non-resistant variants. This study can guide experimentalists to rationally design promising IL–resistant enzymes, and contribute to a deeper understanding of protein–IL interactions at the molecular level