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- 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.
- ItemEnhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization(Weinheim : Wiley-VCH, 2020) Zou, Zhi; Mate, Diana M.; Nöth, Maximilian; Jakob, Felix; Schwaneberg, UlrichStaphylococcus aureus sortase A (SaSrtA) is widely used for site-specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presence of denaturing agents. Loop engineering and subsequent head-to-tail backbone cyclization of SaSrtA yielded the cyclized variant CyM6 that has a 7.5 °C increased melting temperature and up to 4.6-fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6-fold (vs. parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions. © 2020 The Authors. Published by Wiley-VCH GmbH
- ItemEngineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design(Weinheim : Wiley-VCH, 2020) Zhang, Lingling; Cui, Haiyang; Dhoke, Gaurao V.; Zou, Zhi; Sauer, Daniel F.; Davari, Mehdi D.; Schwaneberg, UlrichCopper efflux oxidase (CueO) from Escherichia coli is a special bacterial laccase due to its fifth copper binding site. Herein, it is discovered that the fifth Cu occupancy plays a crucial and favorable role of electron relay in bioelectrocatalytic oxygen reduction. By substituting the residues at the four coordinated positions of the fifth Cu, 11 beneficial variants are identified with ≥2.5-fold increased currents at −250 mV (up to 6.13 mA cm−2). Detailed electrocatalytic characterization suggests the microenvironment of the fifth Cu binding site governs the electrocatalytic current of CueO. Additionally, further electron transfer analysis assisted by molecular dynamics (MD) simulation demonstrates that an increase in localized structural stability and a decrease of distance between the fifth Cu and the T1 Cu are two main factors contributing to the improved kinetics of CueO variants. It may guide a novel way to tailor laccases and perhaps other oxidoreductases for bioelectrocatalytic applications. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
- 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.
- 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.
- ItemDisplay of functional nucleic acid polymerase on Escherichia coli surface and its application in directed polymerase evolution(New York, NY : Wiley, 2020) Chung, Mu-En; Goroncy, Kati; Kolesnikova, Alisa; Schönauer, David; Schwaneberg, UlrichWe report a first of its kind functional cell surface display of nucleic acid polymerase and its directed evolution to efficiently incorporate 2′-O-methyl nucleotide triphosphates (2′-OMe-NTPs). In the development of polymerase cell surface display, two autotransporter proteins (Escherichia coli adhesin involved in diffuse adherence and Pseudomonas aeruginosa esterase A [EstA]) were employed to transport and anchor the 68-kDa Klenow fragment (KF) of E. coli DNA polymerase I on the surface of E. coli. The localization and function of the displayed KF were verified by analysis of cell outer membrane fractions, immunostaining, and fluorometric detection of synthesized DNA products. The EstA cell surface display system was applied to evolve KF for the incorporation of 2′-OMe-NTPs and a KF variant with a 50.7-fold increased ability to successively incorporate 2′-OMe-NTPs was discovered. Expanding the scope of cell-surface displayable proteins to the realm of polymerases provides a novel screening tool for tailoring polymerases to diverse application demands in a polymerase chain reaction and sequencing-based biotechnological and medical applications. Especially, cell surface display enables novel polymerase screening strategies in which the heat-lysis step is bypassed and thus allows the screening of mesophilic polymerases with broad application potentials ranging from diagnostics and DNA sequencing to replication of synthetic genetic polymers. © 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC