How to engineer an organic solvent tolerant lipase: insights from directed evolution and molecular dynamics simulations

Cui, Haiyang; Schwaneberg, Ulrich (Thesis advisor); Elling, Lothar (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


Expanding synthetic capabilities to routinely employ enzymes in organic solvents (OSs) is a dream for protein engineers and synthetic chemists. The beauty of biocatalysis in OSs is because of the promising solvent properties. However, the instability of enzymes in OSs largely limits the expansion of enzymology scope. In this thesis, the question "how to engineer an OS tolerant Bacillus subtilis lipase A (BSLA)" was answered by developing the recombination approaches and revealing the comprehensive enzyme-OS interaction pattern at the molecular level. Meanwhile, the remaining challenges have been solved in directed evolution and molecular dynamics (MD) simulation, such as (i) how to recombine beneficial substitutions efficiently? (ii) the pieces of the puzzle on enzymes’ behavior in OSs.In directed evolution campaign, systematic recombination studies show that poorly performing variants are usually obtained after recombination of 3 to 4 beneficial substitutions. The latter limits researchers in exploiting nature’s potential in generating better enzyme. The Computer-assisted Recombination (CompassR) strategy provides a selection guide for beneficial substitutions that can be recombined to gradually improve enzyme performance by analysis of the relative free energy of folding (ΔΔGfold). The performance of CompassR rule was evaluated by an analysis of 84 recombinants located on 13 positions of BSLA. Furthermore, combining with CompassR, two efficient recombination strategies, named as 2GenReP (two Gene Recombination Process) and InSiReP (In Silico guided Recombination Process), were presented to recombine numerous substitutions efficiently for enhancing the enzyme function. 2GenReP and InSiReP strategies were validated by recombining 15 isolated/clustered BSLA beneficial substitutions. Notably, after screening of ~500 clones, the "best" variant M4 (I12R/Y49R/E65H/N98R/K122E/L124K; identified in both strategies) had remarkable enhanced resistance in 50 % (v/v) 1,4-dioxane (DOX ,14.6-fold), 60 % (v/v) acetone (6.0-fold), 30 % (v/v) ethanol (2.1-fold), and 60 % (v/v) methanol (2.4-fold) compared to BSLA wild type (WT). In essence, CompassR rule, 2GenReP, and InSiReP strategies allow to recombine beneficial substitutions iteratively and empower researchers to generate better enzymes in a time-efficient manner. In MD simulation studies, the reported pieces of the puzzle on the role of OSs on enzymes indicate a need for further studies to solve the OSs puzzle. A comprehensive understanding of such interactions is especially important for protein engineers to design OS resistant enzymes. MD simulation of BSLA WT and BSLA substitutions were performed in OSs, respectively. In terms of BSLA WT, MD simulations showed that three OSs (i.e., DOX, dimethyl sulfoxide (DMSO), 2,2,2-trifluoroethanol (TFE)) reduce BSLA activity and resistance in OSs by (i) stripping off essential water molecules from the BLSA surface mainly through H-bonds binding; and (ii) penetrating the substrate binding cleft leading to inhibition and conformational change. Interestingly, integration of computational results with the "BSLA-SSM" variant library (3439 variants; all-natural diversity with amino acid exchange) revealed two complementary rational design strategies: (i) surface charge engineering, and (ii) non-polar substrate binding cleft engineering. In the case of BSLA substitutions, 20 single substitutions (ten beneficial and ten non-beneficial) were selected for the OS resistance study in TFE. After analyzing 35 possible factors in terms of structure, solvation, and interaction energy at the molecule level, it is found that increased hydration of substituted site is the predominant factor to drive the improved resistance in OS. During the iterative recombination of four beneficial substitutions, molecular hydration extent of BSLA variant correlates positively with their OS resistance (R2 = 0.91), resulting in a super stable BSLA variant (I12R/M137H/N166E) with a 7.8-fold improved OS resistance in 12 % (v/v) TFE. Taking the factors of enzyme hydration as well as surface engineering into account may guide a more effective and efficient route when rationally tailoring enzyme stability in OSs. Overall, the prominent recombination approaches (i.e., CompassR, 2GenReP, and InSiReP) and the rational design strategies (i.e., surface charge and non-polar substrate binding cleft engineering, enzyme hydration guide engineering) achieved to direct researchers design better OS tolerant lipase. The latter rational design strategies were revealed from the molecular understanding of enzyme-OS interaction (i.e., H-bonds binding, hydration effect). Besides, all the discovered design principles are most likely to be transferred to other enzymes sharing a similar α/β-hydrolase fold, which represents an impressive advance in the field of biocatalysis in OSs.