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- ItemEngineering robust cellulases for tailored lignocellulosic degradation cocktails(Basel : MDPI AG, 2020) Contreras, Francisca; Pramanik, Subrata; Rozhkova, Aleksandra M.; Zorov, Ivan N.; Korotkova, Olga; Sinitsyn, Arkady P.; Schwaneberg, Ulrich; Davari, Mehdi D.Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemUnraveling the Mechanism and Kinetics of Binding of an LCI-eGFP-Polymer for Antifouling Coatings(Weinheim : Wiley-VCH, 2021) Söder, Dominik; Garay-Sarmiento, Manuela; Rahimi, Khosrow; Obstals, Fabian; Dedisch, Sarah; Haraszti, Tamás; Davari, Mehdi D.; Jakob, Felix; Heß, Christoph; Schwaneberg, Ulrich; Rodriguez-Emmenegger, CesarThe ability of proteins to adsorb irreversibly onto surfaces opens new possibilities to functionalize biological interfaces. Herein, the mechanism and kinetics of adsorption of protein-polymer macromolecules with the ability to equip surfaces with antifouling properties are investigated. These macromolecules consist of the liquid chromatography peak I peptide from which antifouling polymer brushes are grafted using single electron transfer-living radical polymerization. Surface plasmon resonance spectroscopy reveals an adsorption mechanism that follows a Langmuir-type of binding with a strong binding affinity to gold. X-ray reflectivity supports this by proving that the binding occurs exclusively by the peptide. However, the lateral organization at the surface is directed by the cylindrical eGFP. The antifouling functionality of the unimolecular coatings is confirmed by contact with blood plasma. All coatings reduce the fouling from blood plasma by 8894% with only minor effect of the degree of polymerization for the studied range (DP between 101 and 932). The excellent antifouling properties, combined with the ease of polymerization and the straightforward coating procedure make this a very promising antifouling concept for a multiplicity of applications.
- ItemLess Unfavorable Salt Bridges on the Enzyme Surface Result in More Organic Cosolvent Resistance(Weinheim : Wiley-VCH, 2021) Cui, Haiyang; Eltoukhy, Lobna; Zhang, Lingling; Markel, Ulrich; Jaeger, Karl-Erich; Davari, Mehdi D.; Schwaneberg, UlrichBiocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems. Iterative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance toward three OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and half-life up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50–100 °C. The salt bridge redesign provides protein engineers with a powerful and likely general approach to design OSs- and/or thermal-resistant lipases and other α/β-hydrolases. © 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
- ItemEffects of proline substitutions on the thermostable LOV domain from Chloroflexus aggregans(Basel : MDPI AG, 2020) Remeeva, Alina; Nazarenko, Vera V.; Goncharov, Ivan M.; Yudenko, Anna; Smolentseva, Anastasia; Semenov, Oleg; Kovalev, Kirill; Gülbahar, Cansu; Schwaneberg, Ulrich; Davari, Mehdi D.; Gordeliy, Valentin; Gushchin, IvanLight-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2◦ C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemHow to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study(Weinheim : Wiley-VCH, 2020) Cui, Haiyang; Stadtmüller, Tom H.J.; Jiang, Qianjia; Jaeger, Karl-Erich; Schwaneberg, Ulrich; Davari, Mehdi D.Expanding synthetic capabilities to routinely employ enzymes in organic solvents (OSs) is a dream for protein engineers and synthetic chemists. Despite significant advances in the field of protein engineering, general and transferable design principles to improve the OS resistance of enzymes are poorly understood. Herein, we report a combined computational and directed evolution study of Bacillus subtlis lipase A (BSLA) in three OSs (i. e., 1,4-dioxane, dimethyl sulfoxide, 2,2,2-trifluoroethanol) to devise a rational strategy to guide engineering OS resistant enzymes. Molecular dynamics simulations showed that OSs 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 “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) substrate binding cleft engineering. These strategies are most likely applicable to stabilize other lipases and enzymes and assist experimentalists to design organic solvent resistant enzymes with reduced time and screening effort in lab experiments. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA
- ItemComputer-Assisted Recombination (CompassR) Teaches us How to Recombine Beneficial Substitutions from Directed Evolution Campaigns(Weinheim : Wiley-VCH, 2020) Cui, Haiyang; Cao, Hao; Cai, Haiying; Jaeger, Karl-Erich; Davari, Mehdi D.; Schwaneberg, UlrichA main remaining challenge in protein engineering is how to recombine beneficial substitutions. Systematic recombination studies show that poorly performing variants are usually obtained after recombination of 3 to 4 beneficial substitutions. This limits researchers in exploiting nature's potential in generating better enzymes. 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 was evaluated by analysis of 84 recombinants located on 13 positions of Bacillus subtilis lipase A. The finally obtained variant F17S/V54K/D64N/D91E had a 2.7-fold improved specific activity in 18.3 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). In essence, the deducted CompassR rule allows recombination of beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time-efficient manner. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
- ItemA Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT(Weinheim : Wiley-VCH, 2021) Markel, Ulrich; Lanvers, Pia; Sauer, Daniel F.; Wittwer, Malte; Dhoke, Gaurao V.; Davari, Mehdi D.; Schiffels, Johannes; Schwaneberg, UlrichEnzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases. © 2020 The Authors. Published by Wiley-VCH GmbH
- ItemMatter-tag: A universal immobilization platform for enzymes on polymers, metals, and silicon-based materials(New York, NY : Wiley, 2020) Dedisch, Sarah; Wiens, Annika; Davari, Mehdi D.; Söder, Dominik; Rodriguez-Emmenegger, Cesar; Jakob, Felix; Schwaneberg, UlrichEnzyme immobilization is extensively studied to improve enzyme properties in catalysis and analytical applications. Here, we introduce a simple and versatile enzyme immobilization platform based on adhesion-promoting peptides, namely Matter-tags. Matter-tags immobilize enzymes in an oriented way as a dense monolayer. The immobilization platform was established with three adhesion-promoting peptides; Cecropin A (CecA), liquid chromatography peak I (LCI), and Tachystatin A2 (TA2), that were genetically fused to enhanced green fluorescent protein and to two industrially important enzymes: a phytase (from Yersinia mollaretii) and a cellulase (CelA2 from a metagenomic library). Here, we report a universal and simple Matter-tag–based immobilization platform for enzymes on various materials including polymers (polystyrene, polypropylene, and polyethylene terephthalate), metals (stainless steel and gold), and silicon-based materials (silicon wafer). The Matter-tag–based enzyme immobilization is performed at ambient temperature within minutes (<10 min) in an aqueous solution harboring the phytase or cellulase by immersing the targeted material. The peptide LCI was identified as universal adhesion promoter; LCI immobilized both enzymes on all investigated materials. The attachment of phytase-LCI onto gold was characterized with surface plasmon resonance spectroscopy obtaining a dissociation constant value (KD) of 2.9·10−8 M and a maximal surface coverage of 504 ng/cm². © 2019 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.
- ItemCan constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase(Gotenburg : Research Network of Computational and Structural Biotechnology (RNCSB), 2021) Contreras, Francisca; Nutschel, Christina; Beust, Laura; Davari, Mehdi D.; Gohlke, Holger; Schwaneberg, UlrichCellulases are industrially important enzymes, e.g., in the production of bioethanol, in pulp and paper industry, feedstock, and textile. Thermostability is often a prerequisite for high process stability and improving thermostability without affecting specific activities at lower temperatures is challenging and often time-consuming. Protein engineering strategies that combine experimental and computational are emerging in order to reduce experimental screening efforts and speed up enzyme engineering campaigns. Constraint Network Analysis (CNA) is a promising computational method that identifies beneficial positions in enzymes to improve thermostability. In this study, we compare CNA and directed evolution in the identification of beneficial positions in order to evaluate the potential of CNA in protein engineering campaigns (e.g., in the identification phase of KnowVolution). We engineered the industrially relevant endoglucanase EGLII from Penicillium verruculosum towards increased thermostability. From the CNA approach, six variants were obtained with an up to 2-fold improvement in thermostability. The overall experimental burden was reduced to 40% utilizing the CNA method in comparison to directed evolution. On a variant level, the success rate was similar for both strategies, with 0.27% and 0.18% improved variants in the epPCR and CNA-guided library, respectively. In essence, CNA is an effective method for identification of positions that improve thermostability.
- ItemAqueous ionic liquids redistribute local enzyme stability via long-range perturbation pathways(Gotenburg : Research Network of Computational and Structural Biotechnology (RNCSB), 2021) El Harrar, Till; Frieg, Benedikt; Davari, Mehdi D.; Jaeger, Karl-Erich; Schwaneberg, Ulrich; Gohlke, HolgerIonic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co-)solvents for biocatalysis due to their unique properties. On the other hand, the incubation of enzymes in IL or aIL often reduces enzyme activity. Recent studies proposed various aIL-induced effects to explain the reduction, classified as direct effects, e.g., local dehydration or competitive inhibition, and indirect effects, e.g., structural perturbations or disturbed catalytic site integrity. However, the molecular origin of indirect effects has largely remained elusive. Here we show by multi-μs long molecular dynamics simulations, free energy computations, and rigidity analyses that aIL favorably interact with specific residues of Bacillus subtilis Lipase A (BsLipA) and modify the local structural stability of this model enzyme by inducing long-range perturbations of noncovalent interactions. The perturbations percolate over neighboring residues and eventually affect the catalytic site and the buried protein core. Validation against a complete experimental site saturation mutagenesis library of BsLipA (3620 variants) reveals that the residues of the perturbation pathways are distinguished sequence positions where substitutions highly likely yield significantly improved residual activity. Our results demonstrate that identifying these perturbation pathways and specific IL ion-residue interactions there effectively predicts focused variant libraries with improved aIL tolerance.