Research highlights

 

A competitive high-throughput screening platform for designing polylactic acid-specific binding peptides

Yi Lu Copyright: © Biotec

Lu, Y., Hintzen, K. W., Kurkina, T., Ji, Y., Schwaneberg, U., Advanced Science. https://doi.org/10.1002/advs.202303195.

A 96-well microtiter plate based high-throughput screening system for polylactic acid (PLA) specific binding was developed for detection, sorting, and material-specific degradation of PLA in mixed plastics.

Polylactic acid (PLA) is a rapidly-growing bioplastic with broad applications in food packaging, agricultural films, and drug delivery. To meet the application requirements, PLA is often blended with polypropylene (PP) to enhance its melt processability, thermal stability, and stiffness. The development of plastic recycling processes in the future will be essential to separate PLA from PP in mixed plastics. Material binding peptides (MBPs) have outstanding binding properties to a variety of materials. Engineered MBPs that can bind to polymers in a material-specific manner have a high potential for material-specific detection or enhanced degradation of PLA in mixed PLA/PP plastics. To obtain a material-specific MBP for PLA binding (termed PLAbodies), protein engineering of MBP Cg-Def for improved PLA binding specificity is reported in this work. A 96-well microtiter plate based high-throughput screening system for PLA specific binding (PLABS) was first developed and validated in a protein engineering (KnowVolution) campaign. As a result, a Cg-Def variant V2 (Cg-Def S19K/K10L/N13H) with a 2.3-fold improved PLA binding specificity compared to PP was obtained. Contact angle and surface plasmon resonance measurements confirmed improved material-specific binding of V2 to PLA (1.30-fold improved PLA surface coverage). The established PLABS screening platform for PLA bodies designing can be applied in detection, sorting, and material-specific degradation of PLA in mixed plastics.

Yi Lu is financially supported by the China Scholarship Council (CSC) scholarship. This work was supported by European Union's Horizon 2020 research and innovation program under grant agreement no. 870294 for the project MIX-UP.

To learn more, please access the full paper on Publications and patents and

Lu, Y., Hintzen, K. W., Kurkina, T., Ji, Y., Schwaneberg, U., Advanced Science. https://doi.org/10.1002/advs.202303195.

 
  KnowVolution of Cg-Def Copyright: © Open Access Article
 
 

Enhanced PLA-specific binding of Cg-Def by KnowVolution campaign comprising four phases. In Phase I, potentially beneficial positions were identified by screening cepPCR library of Cg-Def using a 96-well microtiter plate based high-throughput screening system. Then in Phase II, beneficial positions were determined after screening the SSM libraries of identified positions. In Phase III, the computational analysis of determined beneficial positions and substitutions was performed to find compatible substitutions. Finally, a recombined variant was generated and characterized for PLA binding specificity by competitive binding tests in one pot, contact angle, and SPR measurements in Phase IV. PLA: polylactic acid; MBP: material binding peptide; PP: polypropylene; cepPCR: casting error-prone polymerase chain reaction method; SPR: surface plasmon resonance; SSM: site-saturation mutagenesis

 
 

Deep learning guided enzyme engineering of Thermobifida fusca cutinase for increased PET depolymerization

Shuaiqi and Yu Copyright: © Biotec

Meng, S., Li, Z., Zhang, P., Contreras, F., Ji, Y., Schwaneberg, U., Chinese Journal of Catalysis , doi.org/10.1016/S1872-2067(23)64470-5

Applying deep learning for improving enzyme-mediated PET depolymerization efficiency.

A responsible and sustainable circular economy of polymers requires efficient recycling processes with a low CO2 footprint. Enzymatic depolymerization of polyethylene terephthalate (PET) is a first step to make PET polymers a part of a circular economy of polymers. In this study, a structure-based deep learning model was employed to identify residues in TfCut2 that are responsible for improved hydrolytic activity and enhanced stability.

Through the application of machine-learning-guided design, beneficial positions (L32E, S35E, H77Y, R110L, S113E, T237Q, R245Q, and E253H) within the protein structure were identified. These positions were then systematically combined, culminating in the development of the favorable variant, L32E/S113E/T237Q. This variant exhibited improved PET depolymerization when compared with TfCut2 WT (amorphous PET film, 2.9-fold improvement // crystalline PET powder (crystallinity > 40%), 5.3-fold improvement). In terms of thermal resistance, the variant L32E/S113E/T237Q showed a 5.7 °C increased half-inactivation temperature ( T50 60 ). The PET-hydrolysis process was monitored via a quartz crystal microbalance with dissipation monitoring (QCM-D) in real-time to determine depolymerization kinetics of PET coated onto the gold sensor. Finally, conformational dynamics analysis revealed that the substitutions induced a conformational change in the variant L32E/S113E/T237Q, in which the dominant conformation enabled a closer contact between the catalytic site and PET resulting in increased PET-hydrolysis. Overall, this study demonstrates the potential of deep learning models in protein engineering for identifying and designing efficient PET depolymerization enzymes.

Shuaiqi Meng was supported by a Ph.D. scholarship from the China Scholarship Council (CSC No. 201906880011).

To learn more, please access the full paper on publications and patents and

Meng, S., Li, Z., Zhang, P., Contreras, F., Ji, Y., Schwaneberg, U., Chinese Journal of Catalysis , doi.org/10.1016/S1872-2067(23)64470-5

 
 
Deep learning algorithm for engineering TfCut2. Copyright: © Chinese Journal of Catalysis
 
 

Deep learning algorithm was utilized to engineer TfCut2 for a robust and active PET hydrolase, leading to obviously incensement of kinetic stability and activity towards amorphous and crystalline PET.

 
 

Validated High-Throughput Screening System for Directed Evolution of Nylon-Depolymerizing Enzymes

Hendrik Puetz and Christoph Janknecht Copyright: © biotec Hendrik Puetz and Christoph Janknecht

Hendrik Puetz, Christoph Janknecht, Francisca Contreras, Mariia Vorobii, Tetiana Kurkina, and Ulrich Schwaneberg, ACS Sustainable Chemistry & Engineering, doi.org/10.1021/acssuschemeng.3c01575

A sensitive high-throughput screening system enables directed depolymerase evolution to leverage sustainable polyamide and polyurethane recycling.

The accumulation of synthetic polymer waste poses a significant challenge to both society and the environment. Enzymes offer a promising avenue to foster a circular plastic economy by facilitating polymer degradation into monomers, enabling value-preserving and sustainable recycling. Recent years have shown impressive progress in using enzymes to break down PET. However, the lack of efficient enzymes and screening systems for discovering and engineering viable biocatalysts for other major polycondensates like PA and PUR impedes progress in this field. In this work, we report the first highly sensitive colorimetric amine screening system for directed polyamidase and polyurethanase evolution (AMIDE). We used this system to screen the breakdown of PA 6 and PA 6,6 on polymer film and granules and detect common PUR degradation products (2,4-TDA, 2,6-TDA, HMDA, MDA) down to the nanomolar range. We highlighted the applicability of AMIDE for directed depolymerase evolution by kinetically improving the polyamidase NylC. This was achieved through the screening of a small random mutagenesis library. We believe the AMIDE method not only helps to improve existing depolymerases for PA and PUR but also may be applied to screen e.g., metagenomic libraries on authentic polymeric substrates. This could lead to the discovery of novel enzymes to contribute to a circular synthetic polymer economy.

Hendrik Pütz and Christoph Janknecht are financially supported by the German Federal Ministry for Education and Research ([FKZ: 031B0867E], [FKZ: 031B0506C], [FKZ: 031B1342C], [FKZ: 031B1159CX]).

To learn more, please access the full paper on publications and patents and

Puetz, H.; Janknecht, C.; Contreras, F.; Vorobii, M.; Kurkina, T.; Schwaneberg, U. Validated High-Throughput Screening System for Directed Evolution of Nylon-Depolymerizing Enzymes. ACS Sustainable Chemistry & Engineering 2023. doi.org/10.1021/acssuschemeng.3c01575

 
  AMIDE high-throughput screening method Copyright: © ACS Sustainable Chemistry & Engineering
 
 

Figure 1. Synthetic polymer degradation efficiency of enzyme libraries is screened on authentic substrates and released products can be labeled and quantified. This allows the selection of improved enzyme variants in directed polyamidase and polyurethanase evolution campaigns.

 
 

Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s

Shuaiqi and Yu Copyright: © Biotec

Meng, S., Li, Z., Ji, Y., Ruff, A. J., Liu, L., Davari, M. D., Schwaneberg, U., Chinese Journal of Catalysis , doi.org/10.1016/S1872-2067(23)64445-6

Cytochrome P450s are versatile catalysts for biosynthesis applications. In P450s reactions, two electrons are required to reduce the ferric heme iron and activate the subsequent reductions through electron transfer pathways (eTPs), which often represent the rate-limiting step in reactions. Hence, an in-depth understanding of the P450 ET pathway could increase our knowledge of these oxidases and benefit from their further applications.

The electron transfer efficiency of P450s are empowered by introducing aromatic amino acids.

In this study, the P450 BM3 from Bacillus megaterium was engineered for improved catalytic performance by redesigning proposed eTPs. By introducing aromatic amino acids on eTPs of P450 BM3, the “best” variant P2H02 (A399Y/Q403F) showed 13.9-fold improved catalytic efficiency (kcat /KM = 913.5 s-1 M-1 ) compared with P450 BM3 WT. Molecular dynamics simulations and electrons hopping pathways analysis revealed that aromatic amino acid substitutions bridging cofactor flavin mononucleotide and heme iron could increase electron transfer rates and, therefore, improve catalytic performance. Moreover, introducing tyrosines surprisingly showed positive effects on catalytic efficiency by protecting P450s from potential oxidative damage. Furthermore, the variant P2H02 showed improved catalytic efficiency (from 3.8- to 7.1-fold) towards five substrates (indole, benzo-1,4-dioxane, pseudocumene, CTAB, and p-xylene) with varying size and electronic properties. The general applicability of the electron transfer design through aromatic substitutions was also demonstrated by introducing the Q379Y substitution in CYP116B3 (9.16-fold improved catalytic efficiency). In summary, the engineering of eTPs by aromatic amino acid substitutions represents a powerful approach to design catalytically efficient P450s and could be expanded to other oxidases relying on long-range electron transfer pathways. Shuaiqi Meng was supported by a Ph.D. scholarship from the China Scholarship Council (CSC No. 201906880011).

To learn more, please access the full paper on publications and patents and

Meng, S., Li, Z., Ji, Y., Ruff, A. J., Liu, L., Davari, M. D., Schwaneberg, U., Chinese Journal of Catalysis , doi.org/10.1016/S1872-2067(23)64445-6

 
 
  Aromatic amino acid substitutions bridge the cofactor flavin mononucleotide (FMN) and heme, improving electron transfer efficiency and catalytic performance of engineered P450s Copyright: © Chinese Journal of Catalysis
 
 

Aromatic amino acid substitutions bridge the cofactor flavin mononucleotide (FMN) and heme, improving electron transfer efficiency and catalytic performance of engineered P450s.

 
 

A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases

Lilin and Liang Copyright: © biotec

Lilin Feng, Liang Gao, Volkan Besirlioglu, Khalil Essani, Wittwer Malte, Tetiana Kurkina, Yu Ji, and Ulrich Schwaneberg

A versatile and robust flow cytometry-based screening system for the evolution of hydrogen peroxide-producing oxidases

Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re-engineered for synthetic applications. Herein, we developed the versatile and robust flow cytometry-based screening platform “FlOxi” for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe2+ to Fe3+ (Fenton reaction). Fe3+ mediates the immobilization of a His6-tagged eGFP (eGFPHis) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases, a galactose oxidase (GalOx) and a D-amino acid oxidase (D-AAO), yielding a GalOx variant (T521A) with a 4.4-fold lower Km value and a D-AAO variant (L86M/G14/A48/T205) with a 4.2-fold higher kcat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide-producing oxidases and applied for non-fluorescence substrates.

Lilin Feng and Liang Gao are financially supported by a Ph.D. scholarship from the China Scholarship Council (CSC No.201708330279; No.201708330280).

To learn more, please access the full paper on Publications and patents

Lilin Feng, Liang Gao, Volkan Besirlioglu, Khalil Essani, Wittwer Malte, Tetiana Kurkina, Yu Ji, and Ulrich Schwaneberg, Angewandte Chemie International Edition, doi.org/10.1002/ange.202214999

 
  An ultrahigh-throughput screening platform “FlOxi” Copyright: © Angewandte Chemie International Edition
 
 

An ultrahigh-throughput screening platform “FlOxi” was developed by integrating oxidation/Fenton reaction cascade, Fe3+ mediated eGFPHis decoration, and fluorescence-activated-cell-sorting into a general strategy. FlOxi was validated in two evolution campaigns targeting two oxidases, exemplifying the suitability of the platform for screening oxidase variants in an ultrahigh-throughput manner.

 
 

The molecular basis and enzyme engineering strategies for improvement of coupling efficiency in cytochrome P450s

Yu Ji and Shuaiqi Meng in lack-white Copyright: © biotec

Meng, S., Ji, Y., Zhu, L., Dhoke, G. V., Davari, M. D., Schwaneberg, U., Biotechnology Advances, doi.org/10.1016/j.biotechadv.2022.108051

Rational design of efficient cytochrome P450s with high catalytic efficiency and coupling efficiency

Cytochrome P450 (P450s) are the most versatile biocatalysts in nature. The catalytic ability of these hemoproteins has extraordinary potential for the synthesis of pharmaceuticals, plastics and hormones. However, the industrial application of P450s is limited, and one of the main bottlenecks is their low coupling efficiency to non-natural substrates. The undesirable uncoupling reactions result in the extra consumption of expensive cofactor NAD(P)H, and lead to the accumulation of reactive oxygen species and the inactivation of enzymes and organisms. Using protein engineering methods, these limitations can be overcome by engineering and fine-tuning P450s. A systemic perspective of the enzyme structure and the catalytic mechanism is essential for P450 engineering campaigns for higher coupling efficiency. This review provides an overview on factors contributing to uncoupling and protein engineering approaches to minimize uncoupling and thereby generating efficient and robust P450s for industrial use. Contributing uncoupling factors are classified into three main groups: i) substrate binding pocket; ii) ligand access tunnel(s); and iii) electron transfer pathway(s). Furthermore, we draw future directions for combinations of effective state-of-the-art technologies and available software/online tools for P450s engineering campaigns.

Shuaiqi Meng was supported by a Ph.D. scholarship from the China Scholarship Council (CSC No. 201906880011).

To learn more, please access the full paper on publications and patents and

Meng, S., Ji, Y., Zhu, L., Dhoke, G. V., Davari, M. D., Schwaneberg, U., Biotechnology Advances, doi.org/10.1016/j.biotechadv.2022.108051

 
  P450 engineering campaign Copyright: © Biotechnoly Advances
 
 

Uncoupling reactions restrict the biocatalysis process of cytochrome P450s in industrial applications. A comprehensive understanding used for rational designing P450 variants with high coupling efficiency is crucial to P450 engineering campaigns.

 
 

Plastibodies for Multiplexed Detection and Sorting of Microplastic Particles in High-throughput

Wiwik Bauten Copyright: © biotec

Wiwik Bauten, Maximilian Nöth, Tetiana Kurkina, Francisca Contreras, Yu Ji, Cloé Desmet, Miguel-Ángel Serra, Douglas Gilliland, Ulrich Schwaneberg., Science of the Total Environment, doi.org/10.1016/j.scitotenv.2022.160450

Combination of fluorescent polymer-binding peptides (plastibodies) labeling and flow cytometry enables multiplexed quantification and sorting of stained microplastic particles in high-throughput showing high efficiency and accuracy (>90%).

Potential health and ecological risks associated with microplastic contamination demand the development of novel analytics to quantify and sort microplastic particles in aqueous suspensions. Sorting of microplastic would allow subsequent quantification of toxic compounds entrapped in different types of microplastic, through complementary techniques, which would enable the determination of health risks in a material-specific manner. Material-binding peptides (MBPs) like anchor peptides are material-specific and strongly bind to synthetic polymers such as polystyrene, polypropylene, polyethylene terephthalate, and polyurethane, which can be used as a biotechnological tool to tag microplastic. In the present study, we report a method for quantification of microplastic based on MBPs LCIF16C (engineered MBPs Liquid Chromatography Peak I by substituting the amino acid Phe16 with Cys) conjugated to three Alexa-fluorophores (AF488, AF594, and AF647) generating three fluorescent material-binding peptides (also termed plastibodies) (LCIF16C-AF488, LCIF16C-AF594, and LCIF16C-AF647), and followed by Flow cytometry by fluorescence-activated cell sorting (FACS) analysis. The accurate differentiation of labeled and non-labeled particles by flow cytometry (Chi Squared Test, p < 0.01) confirms the chosen labeling strategy efficiency and verifies the power of flow cytometry for the quantification of microplastic in diverse mixtures. Mixtures of polystyrene microplastic that varied in size (500 nm to 5 µm) and varied in labeled populations were analyzed and sorted into distinct populations reaching sorting efficiencies >90 % for 1x106 sorted events. Finally, a multiplexed quantification and sorting with up to three plastibodies was successfully achieved to validate that the combination of plastibodies and flow cytometry is a powerful and generally applicable methodology for multiplexed analysis, quantification, and sorting of microplastic particles.

We gratefully acknowledge the "BioProMare: PLASTISEA" (Harvesting the marine Plastisphere for novel cleaning concepts) project; [FKZ: 031B0867E]) and BioökonomieREVIER_INNO PlastiQuant (FKZ: 031B0918E), the German Federal Ministry of Education and Research (BMBF) for financial support.

To learn more, please access the full paper on Publications and patents and

Wiwik Bauten, Maximilian Nöth, Tetiana Kurkina, Francisca Contreras, Yu Ji, Cloé Desmet, Miguel-Ángel Serra, Douglas Gilliland, Ulrich Schwaneberg., Science of the Total Environment, doi.org/10.1016/j.scitotenv.2022.160450

 
  Schematic illustration of Plastibodies-mediated fluorescent labeling Copyright: © Science of the Total Environment Schematic illustration of Plastibodies-mediated fluorescent labeling enables high-throughput multiplex quantification and sorting of microplastic particles using flow cytometry.
 
 

Enzyme Hydration: How to Retain Resistance in Ionic Liquids

Haiyang Cui. Copyright: © biotec Haiyang "Ocean" Cui

Cui, H., Zhang, L., Eltoukhy, L., Markel, U., Jaeger, K. E., Schwaneberg, U., Davari, M. D., ACS Sustainable Chem. Eng. 2022, 10, 46, 15104–15114

Enzyme hydration determines the resistance of enzyme in ionic liquids

In the past decade, biocatalysis has made great strides. There is a compelling need for finding green and sustainable media for biocatalysis in the industry as the environment can be adversely affected by volatile organic solvents. Ionic liquids (ILs) potentially offer an alternative reaction media for enzyme-catalyzed syntheses with the reduced solvent evaporation and environmental burden, low flammability, and high recoverability. However, ILs as reaction media are often limited by poor enzymatic activity and stability in ILs. We printed a comprehensive IL–enzyme interaction map by studying 45 molecular observables of 30 lipase A from Bacillus subtilis (BSLA) variants in four ILs and a substitutional landscape with 1504 BSLA variants. The results demonstrated that the enzyme hydration shell is the deciding and independent factor determining the enzyme’s IL resistance. A universal positive correlation (up to R2 = 0.96 in 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM][TfO]) and R2 = 0.85 in 1-butyl-3-methylimidazolium chloride ([BMIM]Cl)) was verified, and an experimentally derived ranking of amino acid substitutions is summarized in a list to provide benefits for better protein engineering practice. Hydration-guided engineering yielded a supremely tolerant BSLA variant I12R/D34K/A132K with 8.1-fold, 8.6-fold, 6.6-fold, and 4.6-fold improved tolerance toward [BMIM]Cl, [BMIM]Br, [BMIM]I, and [BMIM][TfO], respectively, when compared to the wild-type BSLA. The obtained knowledge provides a lesson learned on forecasting enzyme stability in ILs and simplifies a rational design of the IL-tolerant enzymes.

Dr. Haiyang Cui was a PhD in Prof. Ulrich Schwaneberg Group in 2016-2020 and now as a postdoc in University of Illinois at Urbana-Champaign, USA (Prof. Huimin Zhao’ group). This work was realized in the division Computational Biology leaded by Dr. Mehdi D. Davari and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0169 and JARA0187).

To learn more, please access the full paper on Publications and patents and

Cui, H., Zhang, L., Yildiz, C.B., Eltoukhy, L., Cheng, L., Jaeger, K.E., Schwaneberg, U. and Davari, M.D., 2022. Enzyme Hydration: How to Retain Resistance in Ionic Liquids. ACS Sustainable Chemistry & Engineering. doi.org/10.1021/acssuschemeng.2c04216

 
  Universal correlation between ionic liquid resistance and hydration. Copyright: © ACS Sustainable Chemistry & Engineering Universal correlation between ionic liquid resistance and hydration and the practical substitution “ranking list” enable us to predict the enzyme stability in ILs and simplify the protocol to design the IL-tolerant enzymes rationally.
 
 

BioAdhere: Tailor-made bioadhesives for epiretinal visual prostheses

Kai Hintzen Copyright: © biotec

Kai-Wolfgang Hintzen, Christian Simons, Kim Schaffrath, Gernot Roessler, Sandra Johnen, Felix Jakob, Peter Walter, Ulrich Schwaneberg, Tibor Lohmann, Biomaterials Science, https://doi.org/10.1039/D1BM01946E

Design and application of biadhesive peptide (peptesive) for fixation of intraocular implants.

Visual prostheses, i.e. epiretinal stimulating arrays, are a promising therapy in treating retinal dystrophies and degenerations. In the wake of a new generation of devices, an innovative method for epiretinal fixation of stimulator arrays is required. We present the development of tailor-made bioadhesive peptides (peptesives) for fixating epiretinal stimulating arrays omitting the use of traumatic retinal tacks. Binding motifs on the stimulating array (poly[chloro-p-xylylene] (Parylene C)) and in the extracellular matrix of the retinal surface (collagens I and IV, laminin, fibronectin) were identified. The anchor peptides cecropin A (CecA), KH1, KH2 (author's initials) and osteopontin (OPN) were genetically fused to reporter proteins to assess their binding behavior to coated microtiter plates via fluorescence-based assays. Domain Z (DZ) of staphylococcal protein A was used as a separator to generate a bioadhesive peptide. Direct and non-direct cytotoxicity testing was performed. Lastly, the fixating capabilities of the peptesives were tested in proof-of-principle experiments. The generation of the bioadhesive peptide required the evaluation of the N- and C-terminal located anchor peptide. The YmPh–CecA construct (N-terminal fusion) showed the highest activity on Parylene C in comparison with the wildtype phytase without the anchor peptide. The C-terminal anchor peptide fusion (eGFP–OPN) was binding to all four investigated ECM proteins. The strongest binding to collagen I was observed for eGFP–KH1, while the strongest binding to fibronectin was observed for eGFP–KH2. The selectivity of binding was checked by incubating eGFP–CecA and eGFP–OPN on ECM proteins and on Parylene C, respectively. Direct and non-direct cytotoxicity testing of the final biadhesive peptide cecropin-A–DZ–OPN showed good biocompatibility properties. Proof-of-concept experiments in post-mortem rabbit eyes suggested an increased adhesion of CecA–DZ–OPN–coated stimulating arrays. This is the first study to prove the applicability and biocompatibility of peptesives for the fixation of macroscopic objects.

The RWTH Aachen University ERS Seed Fund (OPSF473) supported this work.

To learn more, please access the full paper on Publications and patents and

Kai-Wolfgang Hintzen, Christian Simons, Kim Schaffrath, Gernot Roessler, Sandra Johnen, Felix Jakob, Peter Walter, Ulrich Schwaneberg, Tibor Lohmann, Biomaterials Science, https://doi.org/10.1039/D1BM01946E

 
  Concept of the BioAdhere study: A biadhesive peptide (peptesive) CecA-DomainZ-OPN for fixation of an epiretinal stimulator on the retinal surface. Copyright: © Biomaterial Science Concept of the BioAdhere study: A biadhesive peptide (peptesive) CecA-DomainZ-OPN for fixation of an epiretinal stimulator on the retinal surface.
 
 

Rational Design Yields Molecular Insights on Leaf-Binding of Anchor Peptides

Christin Brethauer Copyright: © biotec Christin Brethauer

Molecular dynamics simulations are increasingly being used to study and understand the interactions of atoms and molecules, such as the adhesion of peptides to polymers. While artificial surfaces can be well modeled by such computer simulations, the complex nature and composition of biological surfaces such as plant leaves and fruit surfaces complicate the generation of accurate models.

Here, we present the first detailed three-layered atomistic model of the surface of apple leaves and use it to compute free energy profiles of the adhesion and desorption of APs to and from that surface. Our model is validated by a novel fluorescence-based microtiter plate (MTP) assay that mimics these complex processes and allows for quantifying them. For the anchor peptide (AP) Macaque Histatin, we found that aromatic and positively charged amino acids on one side of the helix majorly contribute to the binding toward the surface wax of apple leaves. The established workflow opens up avenues for further experimental and computational studies revolving around foliar applications, such as optimizing APs and investigating the adsorption, incorporation, or diffusion of herbicides, fungicides, or nutrients on, into, or through the surface wax, cutin, or cellulose. Finally, the generated atomistic models and the MTP assay can be adapted to accommodate different plant types and growing conditions.

This work was realized at the Institute for Pharmaceutical and Medicinal Chemistry of the Heinrich Heine University Düsseldorf, at the Institute of Biotechnology of the RWTH Aachen University, in the DWI – Leibniz Institute for Interactive Materials, in the Department of Ecophysiology and the Institute of Crop Science and Resource Conservation (INRES) of the University of Bonn, in the John von Neumann Institute for Computing (NIC), the Jülich Supercomputing Centre (JSC), in the Institute of Biological Information Processing (IBI-7: Structural Biochemistry), and Institute of Bio- and Geosciences (IBG-4: Bioinformatics) of the Forschungszentrum Jülich GmbH.

To learn more, please access the full paper on Publications and patents and

Dittrich J, Brethauer C, Goncharenko L, Bührmann J, Zeisler-Diehl V, Pariyar S, Jakob F, Kurkina T, Schreiber L, Schwaneberg U, Gohlke H., ACS Appl Mater Interfaces, 2022, doi.org/10.1021/acsami.2c00648

  Workflow for the selection of suitable APs and rational improvement. Copyright: © ACS Appl. Mater. Interfaces Workflow for the selection of suitable APs and rational improvement of their binding properties toward specific plant leaves using a multidisciplinary approach.
 
 

In silico and kinetic modelling of new allosteric inhibitor for human protease ADAM17

Revealing the ADAM17 inhibition mechanisms to give methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity

In Marian's new publication, allosteric inhibition of the human endogenous protease ADAM17 was investigated. A newly found compound (CID17) was previously shown to be an active modulator capable of allosterically regulating the activity of ADAM17. Experimental kinetic modeling as well as computational molecular docking experiments proved the previously hypothesized modulatory inhibitor mechanism. Molecular docking identified the exact binding site of the described exosite inhibitor. Furthermore, the docking results and characterization of the inhibitor serve as a basis for future development of new allosteric inhibitors for ADAM17 in a high-throughput procedure. Thus, the inhibitor found could replace the inhibitors currently used in medicine, which are currently responsible for severe side effects due to their non-specific inhibition of metalloproteases.

This work was performed in the Computational Biology division and was made possible by RWTH Aachen University (ERS Seed Fund Call MScale) and DFG grant DR1013/1 1. The simulations were performed using computational resources provided by JARA-HPC at RWTH Aachen University as part of project RWTH0116.

To learn more, please access the full paper on Publications and patents and

Bienstein M., Minond D., Schwaneberg U., Davari M.D., Yildiz D.; In Silico and Experimental ADAM17 Kinetic Modeling as Basis for Future Screening System for Modulators; IJMS, 2022, DOI:10.3390/ijms23031368

 
  Graphical illustration of ADAM17 Copyright: © International Journal of Molecular Science, MDPI Graphical illustration of ADAM17.
 
 

Polar substitutions on the surface of a lipase substantially improve tolerance in organic solvents

Dr. Haiyang Cui, Markus Vedder, Prof. Dr. Lingling Zhang, Prof. Dr. Karl-Erich Jaeger, Prof. Dr. Ulrich Schwaneberg*, Dr. Mehdi D. Davari*, ChemSusChem, doi.org/10.1002/cssc.202102551

Surface polar engineering is a powerful strategy to generate OS-tolerant lipases and other enzymes

Biocatalysis in organic (co-)solvents (OSs) provides numerous industrially attractive advantages, e.g. the favorable shift of reaction equilibria, increased solubility of substrate/product, alternation of substrate specificity and enantioselectivity, suppression of water-dependent side reactions and easy product recovery. As such, enzymatic reactions conducted in OSs would enable to combine the synthetic power of enzymes with chemical synthesis efficiently in industrial and pharmaceutical fields. However, OSs frequently lead to a dramatic drop in enzymes’ catalytic activity and even deactivation. Herein, we report a comprehensive understanding of interactions between surface polar substitutions and DMSO by integrating the molecular dynamics (MD) simulations of 45 variants from Bacillus subtilis lipase A (BSLA) and substitution landscape in “BSLA-SSM” library. By systematically analyzing 39 structural-, solvation-, and interaction energy-based observables, we discovered hydration shell maintenance, DMSO reduction, and decreased local flexibility simultaneously govern the stability of polar variants in OS. Moreover, the fingerprints of 1644 polar-related variants in three OSs demonstrated that substituting aromatic to polar residue(s) hold great potential to highly improve OSs resistance. Hence, surface polar engineering is a powerful strategy to generate OS-tolerant lipases and other enzymes, thereby adapting the catalyst to the desired reaction and process with OSs. In addition, the decryption of the molecular principles, which lead to the enzyme resistance in OSs, brings substantial knowledge to guide protein engineering campaigns and promote biocatalysis in OSs.

Dr. Haiyang Cui, PhD student (2016-2020) in Prof. Dr. Schwaneberg’ group, was financially supported by the China Scholarship Council (CSC) scholarship. This work was realized in the division Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0169 and JARA0187).

To learn more, please access the full paper on Publications and patents and

Cui, H., Vedder, M., Zhang, L., Jaeger, K. E. Schwaneberg, U, & D. Davari, M. Polar substitutions on the surface of a lipase substantially improve tolerance in organic solvents. ChemSusChem. https://doi.org/10.1002/cssc.202102551

 
  Interactions between surface polar substitutions and DMSO Copyright: © ChemSusChem  

Figure. Investigating the molecular dynamics (MD) simulations of 45 variants from Bacillus subtilis lipase A (BSLA) and 1644 substitutions’ landscape in “BSLA-SSM” library obtained a comprehensive molecular understanding of interactions between surface polar substitutions and DMSO.

 
 

ChemCatChem (1/2022) Cover Feature: Natural Product Diversification by One-Step Biocatalysis using Human P450 3A4

In this study, the synthetic potential of human cytochrome P450 3A4 as a Pichia pastoris-based whole-cell biocatalyst for the diversification of natural products was successfully explored.

In this article, N.D. Fessner et al. investigated the potential of human P450 CYP3A4 for diversification of several compounds belonging to a variety of different natural product classes such as terpenes, alkaloids, stilbenoids, steroids and flavonoids. The human P450 CYP3A4 is expressed in the human liver and plays a decisive role in the body’s detoxification system. It’s responsible for the detoxification of up to 50% of all FDA approved drugs. Efficient expression of this highly promiscuous enzyme in the yeast Komagataella phaffi (Pichia pastoris), produced an efficient whole-cell biocatalyst. 31 different authentic human metabolites were identified, many of them for the first time, and 6 were produced at 100 mg scale. This unprecedented degree of diversification extends the repertoire of efficient enzymatic natural product modification adding a new dimension to an enzyme that has previously been used primarily for inhibition and toxicology studies.

This work has received funding from European Union’s Horizon 2020 research and innovation programme, OXYtrain MSCA-ITN, under grant agreement No 722390. This article was supported by TU Graz Open Access Publishing Fund.

To learn more, please access the full paper on publications and patents and

Fessner, N.D., Grimm, C., Srdič, M., Kroutil, W., Schwaneberg, U., and Glieder, A. (2021), Natural Product Diversification by One-Step Biocatalysis using Human P450 3A4. ChemCatChem, Published manuscript. https://doi.org/10.1002/cctc.202101564

  ChemCatChem Cover Feature (January 2022). Copyright: © ChemCatChem, The European Society of Journal of Catalysis ChemCatChem Cover Feature (January 2022) – accessible at https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cctc.202101843.
 
 

Engineering and emerging applications of artificial metalloenzymes with whole cells

Malte Wittwer#, Ulrich Markel#, Johannes Schiffels, Jun Okuda, Daniel F. Sauer*, Ulrich Schwaneberg*, Nature Catalysis, 2021, 4, 814–827, DOI: 10.1038/s41929-021-00673-3

In this review, we summarize recent applications of artificial metalloenzymes (ArMs) in combination with whole cells and adress approaches to tailor ArMs in whole cells by protein engineering methodologies.

The incorporation of artificial metal cofactors into protein scaffolds leads to a new class of catalysts referred to artificial metalloenzymes (ArMs). Combining ArMs with whole cells creates whole-cell systems with a reaction scope unparalleled in nature. In this review, we summarize recent success stories of ArMs in combination with whole cells in application areas such as biosensing, drug therapy, synthetic biology, and industrial microbiology. We also cover the possibilities to integrate ArMs in whole cells to apply directed-evolution methodologies. In addition, we address the challenges of using ArMs in a whole-cell context, such as catalyst inactivation, and highlight potential developments in the future.

To learn more, please access the full paper on Publications and patents or on the publisher's website.

This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG) through the International Research Training Group ‘Selectivity in Chemo- and Biocatalysis’ (SeleCa) (IRTG 1628) and the Bundesministerium für Bildung und Forschung (BMBF) (FKZ: 031B0297).

  ArM engineering workflow Copyright: © Springer Nature Limited
 
 

Figure. ArM engineering in whole cells allow high-throughput directed-evolution methodologies and can be used to tailor ArMs for specific applications in the fields of drug therapy, biosensing, synthetic biology, and industrial microbiology.

 
 

Kill&Repel Coatings: The Marriage of Antifouling and Bactericidal Properties to Mitigate and Treat Wound Infections

M. Garay-Sarmiento, L. Witzdam, M. Vorobii, C. Simons, N. Herrmann, A. de los Santos Pereira, E. Heine, I. El-Awaad, R. Lütticken, F. Jakob, U. Schwaneberg* and C. Rodriguez-Emmenegger*; Advanced Functional Materials; DOI: 10.1002/adfm.202106656

Wound infections originate when exogenous or endogenous bacterial pathogens can circumvent the barrier of the wound dressing and invade the wound bed. Bacterial colonization causes inflammation, stalls the healing process, and carries the risk of dissemination to other tissues. In addition, current antimicrobial dressings fail to resolve an infection once it has been established because debris of the killed bacteria rapidly accumulates on their surface and hampers the antimicrobial action. Faced with this challenge, hybrid synthetic-natural water-soluble macromolecules are designed that self-assemble onto the surface of dressings to generate an antifouling brush functionalized with endolysin, a bactericidal enzyme that poses no harm for eukaryotic cells. The simultaneous action of the brush and the enzyme not only prevents the colonization of the dressing, but also enables the coating to kill planktonic bacteria with even higher efficiency than the free enzyme. Remarkably, the Kill&Repel coating could completely eradicate bacteria in a simulated infection without allowing the adhesion of residues on the surface. Thus, this strategy opens a revolutionary approach for protecting and treating an infected wound in a safer and more efficient manner.

This work is financially supported by the research association Forschungskuratorium Textil e.V. supported via AiF, research project IGF-No. 19893 N within the promotion program of “Industrielle Gemeinschaftsforschung” (IGF) of the Federal Ministry for Economic Affairs and Energy on the basic of a decision by the German Bundestag. This work was also partially performed at the Center of Chemical Polymer Technology CPT, which was supported by the EU and the federal state of North Rhine-Westphalia (grant EFRE 30 00 883 02).

To learn more, please access the full paper on Publications and patents or on the publisher’s website:

M. Garay-Sarmiento, L. Witzdam, M. Vorobii, C. Simons, N. Herrmann, A. de los Santos Pereira, E. Heine, I. El-Awaad, R. Lütticken, F. Jakob, U. Schwaneberg,* and C. Rodriguez-Emmenegger* DOI: 10.1002/adfm.202106656

  Kill&Repel mechanism Copyright: © Journal of Biotechnology / Elsevier  

Figure. Kill&Repel: LCI-eGFP-Polymer prohibits proteins, skin cells, and bacteria from adhering to the dressing. However, if bacteria surpassed the barrier, the coating is equipped with LCI-EndLys, which hydrolyzes the peptidoglycan of the bacterium upon contact, causing its death. The debris of killed bacteria is then repelled from the surface by LCI-eGFP-Polymer.

 
 

Protein engineering by efficient sequence space exploration through combination of directed evolution and computational design methodologies

A framework of computer-aided protein engineering strategies of (A) FRESCO, (B) FoldX, (C) CNA, (D) PROSS, and (E) ProSAR methods. Copyright: © WILEY‐VCH A framework of computer-aided protein engineering strategies of (A) FRESCO, (B) FoldX, (C) CNA, (D) PROSS, and (E) ProSAR methods.

In this new book chapter, Subrata and co-workers reviewed the current protein engineering strategy by efficient sequence space exploration through a combination of directed evolution and computational design methodologies. The importance of protein engineering for our society was acknowledged by awarding the Noble Prize in Chemistry in 2018 to Frances H. Arnold, George P. Smith, and Sir Gregory P. Winter for their pioneering work on the directed evolution of enzymes and the phage display of peptides and antibodies.

In this book chapter, we highlighted protein engineering strategies, which combine directed evolution and computational methods (e.g., FRESCO, FoldX, CNA, PROSS, ProSAR) to efficiently reengineer enzymes. In this respect, an emphasis was given to the ‘KnowVolution’ strategy, which is generally applicable, minimizes experimental efforts, generates a molecular understanding of each position/amino acid substitution, and was successfully applied to a broad range of enzymes and properties. Finally, we drew attention to a possible future of robust computational design by “in silico” methodologies through the development of quantum computing and machine-learning approaches that hold the promise to fully explore the protein sequence and thereby efficiently use the vast design space that nature offers.

This work was supported through funds from RWTH Aachen University, DWI-Leibniz Institute for Interactive Materials. We gratefully acknowledge the German Federal Ministry of Education and Research (BMBF) (FKZ: 031B0506) for the financial support in the framework of Bioeconomy International. We thank Dr. Daniel Friedrich Sauer for help in preparing the Figure.

To learn more, please access the full paper on publications (https://onlinelibrary.wiley.com/doi/abs/10.1002/9783527815128.ch7) and

Pramanik, S., Contreras, F., Davari, M.D. and Schwaneberg, U. (2021). Protein Engineering by Efficient Sequence Space Exploration Through Combination of Directed Evolution and Computational Design Methodologies. In Protein Engineering (eds H. Zhao, S.Y. Lee, J. Nielsen and G. Stephanopoulos). https://doi.org/10.1002/9783527815128.ch7

 
 

KnowVolution of prodigiosin ligase PigC towards condensation of short-chain prodiginines

KnowVolution of prodigiosin ligase PigC towards condensation of short-chain prodiginines Copyright: © The Royal Society of Chemistry KnowVolution of prodigiosin ligase PigC towards condensation of short-chain prodiginines.

Brands, S., Brass, H. U. C., Klein, A. S., Sikkens, J. G., Davari, M. D., Pietruszka, J., Ruffa, A. J., Schwaneberg, U. (2021). KnowVolution of prodigiosin ligase PigC towards condensation of short-chain prodiginines. Catalysis Science & Technology, 11, 8, 2805-2815, DOI: 10.1039/D0CY02297G

PigC variants from a directed evolution campaign open a biosynthetic route for short-chain prodiginines.

Prodigiosin ligase PigC catalyses the final condensation step in the prodigiosin biosynthesis of Serratia marcescens, in which two pyrrolic precursor molecules are fused linearly to form tripyrrolic red prodiginines. Prodiginine compounds attracted interest as pharmaceutical agents lately since their structural elucidation in the 1960s due to their multiple bioactivities (e.g., antibiotic, nematicidal or anticancer effects). As a key enzyme in the prodigiosin biosynthesis pathway, PigC presents a bottleneck for efficient broad-range prodiginine production because of its limited substrate acceptance. In particular, biosynthesis of short-chain prodiginines is of high synthetic interest due to their effective anticancer properties. In this study, PigC was successfully engineered to efficiently accept short-chain pyrroles following the KnowVolution strategy. Improved variants with up to 10.7-fold increased kcat (7.5 ± 0.4 min−1 compared to 0.7 ± 0.1 min−1 for the PigC wild type), and a nearly 40-fold enhanced catalytic efficiency (kcat/KM = 23.9 ± 3.6 mM−1 s−1 compared to 0.6 ± 0.1 mM−1 s−1 for the PigC wild type) towards short-chain pyrroles were identified. The final and most active PigC variant S1 carried two amino acid substitutions (I365G/M671V) in the substrate-binding domain close to a substrate tunnel, and showed a pronounced preference for small pyrroles. In summary, the directed evolution campaign on PigC provided engineered variants to open a biosynthetic route for short-chain prodiginines.

The Ministry of Culture and Science financially supported the work of Stefanie Brands within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-00213).

To learn more, please access the full paper on Publications and patents and

Brands, S., Brass, H. U. C., Klein, A. S., Sikkens, J. G., Davari, M. D., Pietruszka, J., Ruffa, A. J., Schwaneberg, U. (2021). KnowVolution of prodigiosin ligase PigC towards condensation of short-chain prodiginines. Catalysis Science & Technology, 11, 8, 2805-2815, DOI: 10.1039/D0CY02297G

 
 

Understanding substrate binding and the role of gatekeeping residues in PigC access tunnels

Identification of target residues for semi-rational engineering of PigC access tunnels Copyright: © ChemComm, Royal Society of Chemistry Identification of target residues for semi-rational engineering of PigC access tunnels.

Brands, S., Sikkens, J. G., Davari, M. D., Brass, H. U. C., Klein, A. S., Pietruszka, J., Ruffa, A. J., Schwaneberg, U. (2021). Understanding substrate binding and the role of gatekeeping residues in PigC access tunnels. Chemical Communications, 57, 21, 2681-2684, DOI: 10.1039/d0cc08226k

Molecular understanding of substrate binding and gatekeeping residues of PigC open up the way to pharmaceutically interesting short chain prodiginines.

Prodigiosin ligase PigC catalyses the final step in the prodigiosin biosynthesis of Serratia marcescens. The ATP-dependent condensation leads to the fusion of two pyrrolic prodigiosin precursors, 4-methoxy-2,2′-bipyrrole-5-carbaldehyde and 2-methyl-3-amyl-1H-pyrrole, yielding the red pigment and secondary metabolite prodigiosin. Prodigiosin derivatives form the compound class of prodiginines, which have attracted much attention due to their multiple bioactivities. For instance, effects of prodiginines against plant pathogenic nematodes and fungi indicate their potential use as pesticides in agriculture, while bioactivity against cancer cells holds potential for the pharmaceutical application of prodiginine compounds. Semi-rational redesign of the substrate binding pocket and access tunnels of prodigiosin ligase PigC enhanced the catalytic efficiency in the synthesis of pyrrolic anti-cancer agents more than 45 times. A molecular understanding was gained on residues V333 and T334 relevant to substrate binding and translocation of small pyrroles through PigC access tunnels. In conclusion, the PigC variant V3 (T334A/R674Q) enables synthetic access to pharmaceutically interesting short chain prodiginines (kcat enhanced 3.4 times over the PigC wild type, from 0.9 min-1 to 3.1 min-1). Molecular understanding of the gatekeeper role of residue T334 as a polar barrier in substrate access tunnel 3 and of the key position 333 in the substrate binding pocket further enlightens the PigC catalytic mechanism and empowers rational PigC engineering studies to broaden the substrate profile and enhance the catalytic performance.

The Ministry of Culture and Science financially supported the work of Stefanie Brands within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-00213).

To learn more, please access the full paper on Publications and patents and

Brands, S., Sikkens, J. G., Davari, M. D., Brass, H. U. C., Klein, A. S., Pietruszka, J., Ruffa, A. J., Schwaneberg, U. (2021). Understanding substrate binding and the role of gatekeeping residues in PigC access tunnels. Chemical Communications, 57, 21, 2681-2684, DOI: 10.1039/d0cc08226k

 
 

Progress toward a bicarbonate-based microalgae production system

Mechanism of the bicarbonate pool’s role in the efficient capture of CO2 from the air and rapid carbon supply for photosynthesis. Copyright: © Elsevier Mechanism of the bicarbonate pool’s role in the efficient capture of CO2 from the air and rapid carbon supply for photosynthesis.

Providing efficient and sustainable method to significantly reduce microalgae production cost for their large-scale commercial applications.

Zhu,C.Chen, S., Ji, Y., Schwaneberg, U., Chi, Z., Trends in Biotechnology, doi.org/10.1016/j.tibtech.2021.06.005

Sustainable development of human society strongly depends upon efficient production of biomass, and exploiting untapped opportunity for primary production of biomass is one of the few options to support sustainable development. One promising resource is microalgae, which hold significant advantages over terrestrial crops in terms of their high productivity, high content of target products, ability to adapt to harsh environments, and minimal land use by using ocean space. Microalgae also have other great potential applications on CO2 fixation, treatment of wastewater and biodegradation of plastics. However, these commercial applications are seriously hampered by the high production costs of microalgae, and conventional carbon supplies approach is one of the key reasons.

With using high concentration bicarbonate as carbon source, the bicarbonate-based carbon capture and algae production system (BICCAPS) is a promising solution to significantly reduce microalgae production cost, due to its potential advantages in carbon supply, PBR development, biomass harvesting, as well as labor and energy saving. This review comprehensively summarized these advantages, as well as their recent progress, validating their potential to substantially reduce microalgae production cost. The draw-backs and challenges in improving performance of the BICCAPS was also critically evaluated, and the potential solutions for these problems were discussed. Finally, a perspective was presented for future research efforts on further improving production efficiency and reducing energy inputs for the BICCAPS. This work is very interesting and a valuable contribution to microalgal biotechnology, and provides a practical solution to go carbon neutral by using microalgae.

Dr. Chenba Zhu works in the subgroup “PlastiQuant” and focuses on microplastic degradation by microalgae. This work was supported by the National Natural Science Foundation of China (41861124004), Fundamental Research Funds for the Central Universities [DUT18RC(3)041], Major and Special Program on Science and Technology Projects in Dalian City (2020ZD23SN009) and China Postdoctoral Science Foundation (2019M650054).

To learn more, please access the full paper on Publications and patents and

Zhu,C.Chen, S., Ji, Y., Schwaneberg, U., Chi, Z., Trends in Biotechnology, doi.org/10.1016/j.tibtech.2021.06.005.

 
 

Enzyme mimetic microgel coating for endogenous nitric oxide mediated inhibition of platelet activation

Microgel coating on a membrane Copyright: © Elsevier Inc. Enzyme mimetic microgel coating for the inhibition of platelet activation

Hosseinnejad A., Fischer T., Jain P., Bleilevens C., Jakob F., Schwaneberg U., Rossaint R., Singh S., Journal of Colloid and Interface Science, doi.org/10.1016/j.jcis.2021.05.143

An antifouling microgel coating that mimics glutathione peroxidase inhibits platelet activation and prolongs blood clotting time.

Nitric oxide (NO) continuously generated by healthy endothelium prevents platelet activation and maintains vascular homeostasis. However, when artificial surfaces come in contact with blood, protein adsorption and platelet activation occur, eventually leading to thrombus formation. To overcome this, we present an antifouling microgel coating mimicking the function of the enzyme glutathione peroxidase to endogenously generate NO in the blood plasma from endogenous NO-donors and maintain a physiological NO flux. Microgels are synthesized by copolymerization of highly hydrophilic N–(2–hydroxypropyl)methacrylamide (HPMA) and glycidyl methacrylate (GMA) with diselenide crosslinks. Bioengineered amphiphilic anchor peptides with free thiols are used to immobilize the microgels on hydrophobic poly(4-methylpentene) (TPX) membranes. The hydrophilic nature of the microgel coating prevents protein adsorption while the reversible diselenide bridges make the microgels responsive to the reducing environment and lead to the formation of reactive selenols/selenolates. This subsequently provides an efficient and sustained NO-release from endogenous S-nitrosothiols (RSNOs), mimicking the enzymatic function of glutathione peroxidase. On exposure to the whole blood, the microgel coating inhibited platelet activation and prolonged the blood clotting time.

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) via Schwerpunktprogramm ‘‘Towards an Implantable Lung” (Project number: 347367912 and RO 2000/25-1.)

To learn more, please access the full paper on Publications and Patents and

Hosseinnejad A., Fischer T., Jain P., Bleilevens C., Jakob F., Schwaneberg U., Rossaint R., Singh S., Journal of Colloid and Interface Science, doi.org/10.1016/j.jcis.2021.05.143

 
 

The molecular basis of spectral tuning in blue- and red-shifted Flavin-binding fluorescent proteins

Röllen, K., Granzin, J., Remeeva A., Davari, M. D., Gensch, T., Nazarenko V. V., Kovalev, K., Bogorodskiy, A., Borshchevskiy, V., Hemmer, S., Schwaneberg, U., Gordeliy, V., Jaeger, K. E., Batra-Safferling, R., Gushchin, I., Krauss, U., Journal of Biological Chemistry, doi.org/10.1016/j.jbc.2021.100662

Blue- and red-shifted Flavin-binding fluorescent protein variants represent promising tools for applications in life sciences.

Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plant-derived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a red-shifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences.

This work was financially supported by Deutsche Forschungsgemeinschaft (DFG) Training Group 1166 “BioNoCo” (Biocatalysis in Non-conventional Media), and realized in collaboration with the Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, IBI-7: Structural Biochemistry, Forschungszentrum Jülich GmbH, Jülich, Germany; JuStruct: Jülich Center for Structural Biology, Forschungszentrum Jülich, Jülich, Germany; Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia; Institute of Biotechnology, RWTH Aachen University, Aachen, Germany; IBI-1: Molecular and Cellular Physiology, Forschungszentrum Jülich GmbH, Jülich, Germany; Institut de Biologie Structurale Jean-Pierre Ebel, Université Grenoble Alpes-Commissariat à l’Energie Atomique et aux Energies Alternatives–CNRS, Grenoble, France; Institute of Crystallography, RWTH Aachen University, Aachen, Germany; IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany; DWI-Leibniz Institute for Interactive Materials, Aachen, Germany. The computing resources of this work was granted by JARA-HPC from RWTH Aachen University (JARA0065).

To learn more, please access the full paper on Publications and Patents and Röllen, K., Granzin, J., Remeeva A., Davari, M. D., Gensch, T., Nazarenko V. V., Kovalev, K., Bogorodskiy, A., Borshchevskiy, V., Hemmer, S., Schwaneberg, U., Gordeliy, V., Jaeger, K. E., Batra-Safferling, R., Gushchin, I., Krauss, U., Journal of Biological Chemistry, doi.org/10.1016/j.jbc.2021.100662

 
 

Chemogenetic engineering of nitrobindin toward an artificial epoxygenase

Daniel Sauer and Malte Wittwer Copyright: © Bio VI Daniel Sauer and Malte Wittwer

Sauer D. F., Wittwer M., Markel U., Minges A., Spiertz M., Schiffels J., Davari M. D., Groth G., Okuda J., and Schwaneberg U., Catalysis Science & Technology, doi.org/10.1039/D1CY00609F

Chemogenetic approach and directed evolution enabled the conversion of a natural metalloprotein into an artificial metalloenzyme with >7-fold increase in activity.

Chemogenetic engineering of metalloproteins emerges as a powerful strategy to generate proteins that catalyze non-natural reactions or convert non-natural substrates. Here, we report an artificial metalloenzyme based on the β-barrel protein nitrobindin (NB) equipped with a manganese protoporphyrin IX catalyst (MnPPIX@NB) for the epoxidation of aromatic alkenes. After exchanging the metal cofactor and two rounds of directed evolution, our artificial metalloenzyme shows improved activity (>7-fold increase) with an enantiomeric excess of 20%. The evolution campaign also revealed the importance of the proximal ligand for peroxide activation and subsequent oxygen atom transfer. By utilizing cell adhesion-promoting peptides, a facile strategy is presented to immobilize artificial metalloenzymes on the surface of E. coli cells for on-cell catalysis and chemogenetic engineering of artificial metalloenzymes. We envision that adhesion-peptide-based cell surface functionalization has enormous potential to include artificial enzymes in whole-cell cascade reactions and on-cell catalytic applications.

This work was realized in the division Computational Biology and was funded by the Bundesministerium für Bildung und Forschung (FKZ 031B0297). The support and infrastructure provided by the “Center for Structural Studies” (CSS) at the Heinrich Heine University Düsseldorf, was funded by the Deutsche Forschungsgemeinschaft (DFG grant number 417919780, INST 208/740-1 FUGG). Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under the project JARA0065.

To learn more, please access the full paper on Publications and Patents and

Sauer D. F., Wittwer M., Markel U., Minges A., Spiertz M., Schiffels J., Davari M. D., Groth G., Okuda J., and Schwaneberg U., Catalysis Science & Technology, doi.org/10.1039/D1CY00609F

  Chemical and protein engineering of nitrobindin wildtype Copyright: © Royal Society of Chemistry Chemical and protein engineering of nitrobindin wildtype
 
 

CompassR-guided recombination unlocks design principles to stabilize a lipase in ILs with minimal experimental efforts

Haiyang Cui Copyright: © Bio VI Haiyang Cui

Cui, H., Pramanik S., Jaeger, K. E., Davari, M. D., Schwaneberg, U., Green Chemistry, doi.org/10.1039/D1GC00763G

CompassR analysis and computation-assisted selection enabled, with minimal experimental efforts, to significantly improve BLSA resistance toward four ionic liquids.

Biocatalysis in ionic liquids has gained enormous attention for the production of biodiesel, sugar esters, and pharmaceuticals. However, hydrophilic ionic liquid interaction with enzymes often results in reduced activity or even inactivation. In this report, we prove that intrinsic lipase stability and preservation of hydration shells of Bacillus subtilis lipase A (BSLA) are two synergistic design principles to retain enzymatic activity in ILs. After in silico screening of nine beneficial amino acid positions by the CompassR rule (in total, 172 variants), we rationally designed two variants, to be constructed by site-directed mutagenesis, and three libraries by site-saturation mutagenesis. With minimal experimental effort, we identified three all-around variants towards four 1-butyl-3-methylimidazolium (BMIM)-based ionic liquids. Computational analysis of molecular dynamics and thermodynamic stability analysis of the variants revealed the molecular basis for the resistant variants as the synergistic enhancement of protein stability and increased hydration shells around the substitutions in the four ionic liquids. These design principles and the gained molecular knowledge not only open the door to direct experimentalists for rationally designing promising ionic liquid-resistant enzymes, but also provide new insights into enzymatic catalysis in ionic liquids.

Haiyang Cui is financially supported by the China Scholarship Council (CSC) scholarship. This work was realized in the division Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0169 and JARA0187).

To learn more, please access the full paper on Publications and Patents and

Cui, H., Pramanik S., Jaeger, K. E., Davari, M. D., Schwaneberg, U., Green Chemistry, doi.org/10.1039/D1GC00763G

  Functionality of CompassR Copyright: © Royal Society of Chemistry CompassR-guided recombination
 
 

Fe(III)-complex mediated bacterial cell surface immobilization of eGFP and enzymes

Lilin Feng and Liang Gao Copyright: © BIO VI Lilin Feng and Liang Gao

Feng, L., Gao, L., Sauer, D. F., Ji, Y., Cui, H., Schwaneberg, U. (2021). Fe(III)-complex mediated bacterial cell surface immobilization of eGFP and enzymes. ChemComm, DOI: 10.1039/d1cc01575c

A facile and reversible method to immobilize a broad range of His6-tagged proteins on the E. coli cell surface through Fe (III)–metal complexes

Bacterial cell surface decoration with proteins is highly attractive for biotechnological process and chemical production. Herein, we report a facile, non-covalent and reversible method to immobilize a broad range of His6-tagged proteins on the surface of E. coli cell through Fe (III)-metal complexes. As a proof of principle, His6-tagged proteins were successfully immobilized on E. coli cell surface: Bacillus subtilis lipase A (BSLA), Candida tropicalis fatty alcohol oxidase (CtFAO), Bacillus licheniformis laccase (BlcotA), Armoracia rusticana horseradish peroxidase (ArHRP) and eGFP. Functionality of the ArHRP was additionally demonstrated by generating a hydrogel around E. coli cells. Being able to immobilize eGFP, four enzymes and generating a hydrogel shell around E. coli prove the versatility of metal complex-based immobilization with broad application potentials in catalysis and biosensing.

Lilin Feng and Liang Gao are supported by PhD scholarships from the China Scholarship Council (CSC No. 201708330279; No. 201708330280). This research was funded by the ‘‘Bundesministerium für Bildung und Forschung’’ (BMBF) (FKZ: 031B0297).

To learn more, please access the full paper on Publications and patents (Link) and

Feng, L., Gao, L., Sauer, D. F., Ji, Y., Cui, H., Schwaneberg, U. (2021). Fe(III)-complex mediated bacterial cell surface immobilization of eGFP and enzymes. ChemComm, DOI: 10.1039/d1cc01575c

  Immobilizing proteins through Fe (III)-metal complexes Copyright: © Chemical Communications A facile, non-covalent and reversible method to immobilize a broad range of His6-tagged proteins on the surface of E. coli cell through Fe (III)-metal complexes.
 
 

Chemogenetic Evolution of a Peroxidase-like Artificial Metalloenzyme

Uli Markel,_Daniel Sauer,_Malte Wittwer Copyright: © BIO VI

Ulrich Markel, Daniel F. Sauer, Malte Wittwer, Johannes Schiffels, Haiyang Cui, Mehdi D. Davari, Konstantin W. Kröckert, Sonja Herres-Pawlis, Jun Okuda, Ulrich Schwaneberg, ACS Catalysis, 2021, DOI: 10.1021/acscatal.1c00134.

Chemogenetic evolution led to an artificial peroxidase-like metalloenzyme.

Artificial metalloenzymes consist of a synthetic cofactor embedded in a protein scaffold and thus provide a clean slate for directed evolution studies. Here, the design and directed evolution of an artificial metalloenzyme with peroxidase-like properties was reported. Based on the nitrobindin variant NB4, different porphyrin derivatives were screened in the first round of our chemogenetic evolution campaign. After identifying MnPPIX as the most suitable cofactor, two further rounds of directed evolution increased the enzyme’s activity to levels on par with some natural peroxidases. The introduction of an arginine residue in a position distal to the cofactor was responsible for the increased activity. Furthermore, the NB4 environment resulted in a more flexible distal arginine compared to the respective wildtype-derived variant. The evolved artificial enzyme efficiently oxidized common redox mediators (ABTS, syringaldehyde, and 2,6-dimethoxyphenol) and readily decolorized three recalcitrant dyes (indigo carmine, reactive blue 19, and reactive black 5). Finally, the recycling of the artificial enzyme was demonstrated.

We gratefully acknowledge financial support from the Bundesministerium für Bildung und Forschung (BMBF)(FKZ 031B0297).

To learn more, please access the full paper on Publications and patents and

Ulrich Markel, Daniel F. Sauer, Malte Wittwer, Johannes Schiffels, Haiyang Cui, Mehdi D. Davari, Konstantin W. Kröckert, Sonja Herres-Pawlis, Jun Okuda, Ulrich Schwaneberg, ACS Catalysis, 2021, DOI: 10.1021/acscatal.1c00134.

  Scheme: Chemogenetic evolution of an artificial peroxidase-like metalloenzyme. Copyright: © ACS Catalysis Scheme: Chemogenetic evolution of an artificial peroxidase-like metalloenzyme.
 
 

Less unfavorable salt bridges on the enzyme surface result in more organic cosolvent resistance

Haiyang "Ocean" Cui Copyright: © Bio VI

Cui, H., Eltoukhy, L., Zhang, L., Markel, U., Jaeger, K. E., Davari, M. D., Schwaneberg, U., Angewandte Chemie, doi.org/10.1002/ange.202101642

Removing unfavorable surface salt bridges increased the organic solvent and thermal resistance of enzymes.

The application of organic (co-)solvents (OSs) as reaction media for biocatalysts is mandatory for a large number of applications in the chemical industries. OSs are needed to solubilize hydrophobic substrates and products, they allow for easy product recovery and shift the reaction equilibrium into the desired direction. However, native enzymes often suffer from low activity and/or sensitivity in the presence of OSs, constraining their extended application for biocatalysis. 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.

Haiyang Cui is financially supported by the China Scholarship Council (CSC) scholarship. This work was realized in the division Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0169 and JARA0187).

To learn more, please access the full paper on Publications and patents and

Cui, H., Eltoukhy, L., Zhang, L., Markel, U., Jaeger, K. E., Davari, M. D., Schwaneberg, U., Angewandte Chemie International Edition, doi.org/10.1002/ange.202101642

  Rational salt bridge  design strategy: Enzyme surface interacts with organic solvents. Copyright: © Angewandte Chemie International Edition By removing unfavorable surface salt bridges, the organic solvent and thermal resistance of enzymes can be significantly improved. This design strategy results in locally refined flexibility and strengthened hydration.
 
 

Rapid and Oriented Immobilization of Laccases on Electrodes via a Methionine-Rich Peptide

Haiyang Cui and Lingling Zhang Copyright: © Bio VI Haiyang Cui (l.) and Lingling Zhang (r.)

Haiyang Cui, Lingling Zhang*, Dominik Söder, Xiaomei Tang, Mehdi D. Davari, and Ulrich Schwaneberg*. ACS Catalysis, 2021. 11, 4, 2445-2453.

MetRich is a valuable binding motif for the rapid immobilization and high performance of laccases and other oxidoreductases in bioelectrocatalytic applications.

Nowadays, enzymatic bioelectrocatalysis has found wide applications ranging from electrochemical biosensing platforms, implantable enzymatic fuel cells, to bioelectrosynthetic reactors. Nonetheless, insufficient electron-transfer rates and long-term instabilities of enzyme electrodes are still challenging issues. Herein, we reveal that MetRich (methionine-rich segment) plays an important role in rapid immobilization of copper efflux oxidase (CueO from Escherichia coli) on electrodes by studying the adsorption and bioelectrocatalysis behavior of CueO, a truncated CueO (ΔMetRich CueO), and a serine-rich substituted CueO (SerRich CueO). Atomic molecular dynamics (MD) simulations demonstrate that the synergistic effect of π–π stacking and hydrophobic interactions contribute to the high affinity of MetRich to carbon nanotubes (CNT). To demonstrate the broad applicability, MetRich was also fused to spore coat protein A (CotA), another bacterial laccase from Bacillus licheniformics. It is known that the T1 Cu active site of CotA is around the C-terminus and the entry site of electrons in the laccase-catalyzed oxygen reduction. Fusion of MetRich to the C-terminus of CotA is found to endow CotA with the properties of rapid and oriented adsorption at the electrode surface, “draws” the T1 Cu active site close to the CNT electrode, and thereby increases the electron-transfer rate. The finding and validation make MetRich a valuable binding motif for the rapid immobilization and high performance of laccases and perhaps other oxidoreductases in bioelectrocatalytic applications.

L. Zhang acknowledges the support from the Alexander von Humboldt Foundation. Haiyang Cui is financially supported by the China Scholarship Council (CSC) scholarship. This work was realized in the division Hybrid Catalysis and High Throughput Screening and was supported by division Computational Biology and computing resources granted by JARA-HPC from RWTH Aachen University (JARA0169 and JARA0187).

To learn more, please access the full paper on Publications and patents and

Cui, H.; Zhang, L.*; Söder, D.; Tang, X. M.; Davari, M. D.; Schwaneberg, U.*, Rapid and oriented immobilization of laccases on electrodes via a methionine-rich peptide. ACS Catalysis, 2021. 11, 4, 2445-2453. https://doi.org/10.1021/acscatal.0c05490

  Immobilisation through the MetRich binding motif. Copyright: © ACS Catalysis A valuable binding motif MetRich can drive laccases and other oxidoreductases to the electrode surface (e.g., CNT, Au) with rapid and oriented immobilization.
 
 

Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase

Francisca Contreras and Christina Nutschel Copyright: © BIO VI Francisca (l.) and Christina (r.)

Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo- β -glucanase

Contreras, F.*, Nutschel, C.*, Beust, L., Davari, M. D., Gohlke, H., & Schwaneberg, U., Computational and Structural Biotechnology Journal, 2021, Volume 19, 743-751. DOI 10.1016/j.csbj.2020.12.034
*These authors contributed equally to the work.

Die „Constraint Network Analysis (CNA)“ ist eine vielversprechende Methode zur Identifizierung von vorteilhaften Positionen in Phase I einer KnowVolution-Kampagne zur Verbesserung der Thermostabilität.

Projekt und Förderung: Maßgeschneiderte Enzymcocktails für den effizienten Celluloseabbau (EnzyBioDeg), BMBF, FKZ: 031B0506

Dazugehörige Veröffentlichung: KnowVolution of a GH5 cellulase from Penicillium verruculosum to improve thermal stability for biomass degradation

Abteilung: Computational Biology

Das Protein-Engineering hat sich als ein wichtiges Werkzeug zur Verbesserung der Thermostabilität von Enzymen herausgestellt. Da gerichtete Evolutionskampagnen noch immer mit hohen Zeit- und Arbeitsaufwand verbunden sind, stellt die effiziente Entwicklung von robusten Biokatalysatoren heutzutage noch eine große Herausforderung dar. Kombinierte Strategien aus gerichteter Evolution und computergestützten Analysen stellen daher einen effizienteren Ansatz für das Protein-Engineering dar. Die KnowVolution-Protein-Engineering-Strategie kombiniert gerichtete Evolution und computergestützte Analysen, um den experimentellen Aufwand zu verringern und die Verbesserungen der Enzymvarianten zu maximieren. In "Phase I: Identifizierung" wird die Zufallsmutagenese verwendet, um vorteilhafte Positionen in einem Enzym zu identifizieren. Eine kombinierte Strategie aus einer in silico Positionsauswahl, gefolgt von einer Durchmusterung einer Variantenbibliothek, kann den gesamten experimentellen Aufwand für die Verbesserung eines Biokatalysators reduzieren.

In dieser Studie untersuchen wir die „Constraint Network Analysis“ (CNA)-Strategie als computergestützte Methode, um vorteilhafte Positionen für eine verbesserte Thermostabilität der industriell wichtigen Endo-β-Glucanase Cel5A aus Penicillium verruculosum zu identifizieren. Zusätzlich haben wir das Potenzial von CNA in Protein-Engineering-Kampagnen (z. B. in der Identifikationsphase von KnowVolution) evaluiert. Mit der CNA-Methode kann der Screening-Aufwand um 40 % verringert werden. Eine zufallsmutagenisierte Bibliothek erforderte das Screening von ~8000 Klonen, wobei nur 0,27 % der Klone eine erhöhte Thermostabilität aufwiesen. Die CNA-geführte Bibliothek bestand aus 3840 Klonen und besaß eine vergleichbare Erfolgsrate von 0,18 %. Die Abdeckung der identifizierten Positionen, die die Thermostabilität verbessern, betrug 8 % für die CNA-Methode und 5,7 % für die Zufallsmutagenese. Wir glauben, dass die CNA-Methode in der Identifikationsphase einer KnowVolution-Kampagne eingesetzt werden kann, um die Thermostabilität von Cellulasen und anderen Enzymen mit geringerem Zeit- und Arbeitsaufwand zu verbessern.

  Utilization of Constraint Network Analysis or random mutagenesis in the identification Phase of a KnowVolution engineering campaign. Copyright: Computational and Structural Biotechnology. Copyright: © Computational and Structural Biotechnology Journal Figure. Utilization of Constraint Network Analysis or random mutagenesis in the identification Phase of a KnowVolution engineering campaign. Copyright: Computational and Structural Biotechnology.
 
 

Enzyme Hydration Determines Resistance in Organic Cosolvents

Haiyang "Ocean" Cui Copyright: © BIO VI

Cui, H., Zhang, L., Eltoukhy, L., Jiang, Q., Korkunç, S. K., Jaeger, K. E., Schwaneberg, U., Davari, M. D. ACS Catalysis, 2020. 10, 14847-14856.

Increased enzyme surface hydration of substituted sites as the predominant factor to drive the improved resistance in organic cosolvents

Organic solvents (OSs) are attractive in biocatalysis as they offer numerous advantages, such as increased solubility of substrates/products or suppression of side reactions. However, the use of enzymes in OSs often correlates with enzyme deactivation or a dramatic drop in catalytic activity. In this study, we have developed a comprehensive understanding of enzymes and OSs interactions based on 35 observables obtained from molecular dynamics simulation of 32 resistant and nonresistant Bacillus subtilis lipase A (BSLA) variants (wild type, single, and multiple substitutions) towards an organic cosolvent (12 % (v/v) TFE). Detailed analysis of the distribution of these structural and dynamics observables uncovered that increased enzyme surface hydration of substituted sites as the predominant factor to drive the improved resistance in OS. Furthermore, the iterative recombination of four surface substitutions revealed that the extent of hydration in BSLA variants correlates strongly with its OS resistance (= 0.91). Remarkably, the recombination of substitutions led to a highly resistant BSLA variant (I12R/M137H/N166E) with a 7.8-fold improved resistance in 12 % (v/v) TFE, while retaining comparable catalytic activity (~92%) compared to wild type. Our findings confirm that strengthening protein surface hydration via surface charge engineering is an effective and efficient rational strategy for tailoring of enzyme stability in OSs.

Haiyang Cui is financially supported by the China Scholarship Council (CSC) scholarship. This work was realized in the division Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0169 and JARA0187).

To learn more, please access the full paper on Publications and patents and

Cui, H., Zhang, L., Eltoukhy, L., Jiang, Q., Korkunç, S. K., Jaeger, K. E., Schwaneberg, U., Davari, M. D. (2020). Enzyme Hydration Determines Resistance in Organic Cosolvents. ACS Catalysis, 10, 14847-14856. https://doi.org/10.1021/acscatal.0c03233

  Hydration-guided protein engineering Copyright: © ACS Catalysis Strengthening protein surface hydration through surface charge engineering is an effective and efficient strategy for rational tailoring of enzyme stability in organic solvents.
 
 

MicroGelzymes: pH-Independent Immobilization of Cytochrome P450 BM3 in Microgels

Maximilian Nöth and Larissa Hussmann Copyright: © BIO VI

Maximilian Nöth, Larissa Hussman, Thomke Belthle, Islam El-Awaad, Mehdi D. Davari, Felix Jakob, Andrij Pich*, and Ulrich Schwaneberg* Biomacromolecules, 2020, DOI: doi.org/10.1021/acs.biomac.0c01262

‡ These authors contributed equally to the work

Project: SFB 985

Funding: DFG: Collaborative Research Centre 985 “Functional Microgels and Microgel Systems; BMBF: Next Generation of Biotechnological Processes – Biotechnology 2020+

Subgroup: Biohybrid Systems and Computational Biology

 

The electrostatic immobilization of cytochrome P450 BM3 monooxygenase was achieved in permanently positively charged poly(N-vinylcaprolactam) microgels with 1-vinyl-3-methylimidazolium as comonomer without loss of catalytic activity.

Microgels are an emerging class of enzyme carriers due to their chemical and process stability, biocompatibility, and high enzyme loading capability. In this work, we synthesized a new type of permanently positively charged poly(N-vinylcaprolactam) microgel with 1-vinyl-3-methylimidazolium (quaternization of nitrogen by methylation of N-vinylimidazole moieties) as comonomer (PVCL/VimQ) through precipitation polymerization. The PVCL/VimQ microgels were characterized with respect to their size, charge, swelling degree, and temperature responsiveness in aqueous solutions. Cytochrome P450 monooxygenases are usually challenging to immobilize, and often high activity losses occur after the immobilization (in the case of P450 BM3 up to 100% loss of activity). The electrostatic immobilization of P450 BM3 in permanently positively charged PVCL/VimQ microgels (P450 µ-Gelzymes) was achieved without loss of catalytic activity at the pH optimum of P450 BM3. In addition, P450 µ-Gelzymes could be employed for reversible ionic strength-triggered release and re-immobilization of P450 BM3 as well as catalyst recycling for multiple reuse cycles. Finally, a characterization of the potential of P450 µ-Gelzymes to provide resistance against organic cosolvents (acetonitrile, dimethyl sulfoxide, 2-propanol) was performed to evaluate the biocatalytic application potential of P450 µ-Gelzymes.

  pH-independent electrostatic immobilization of P450 BM3 Copyright: © American Chemical Society  

Figure The pH-independent electrostatic immobilization of P450 BM3 could be achieved in permanently positively charged poly(N-vinylcaprolactam) microgels with 1-vinyl-3-methylimidazolium as comonomer. The permanent positive charge was introduced by quaternization of 1-vinylimidazole through methylation of N-vinylimidazole moieties. Reproduced with permission from Nöth et al. (2020).

 
 

Biocatalytic microgels (μ-Gelzymes): synthesis, concepts, and emerging applications

Elisabeth Gau and Maximilian Noeth Copyright: © BIO VI

Maximilian Nöth, Elisabeth Gau, Falco Jung, Mehdi D. Davari, Islam El-Awaad,* Andrij Pich,* and Ulrich Schwaneberg* Green Chemistry, 2020, DOI 10.1039/D0GC03229H

These authors contributed equally to the work

Project: SFB 985

Funding: DFG: Collaborative Research Centre 985 “Functional Microgels and Microgel Systems; BMBF: Next Generation of Biotechnological Processes – Biotechnology 2020+

Subgroup: Biohybrid Systems and Computational Biology

 

Enzymes are nature’s catalysts able to perform (bio)chemical reactions with impressive chemo-, regio-, and stereoselectivities and have great potential for sustainable processes from renewable resources. The adaptation of enzymes to the requirements of industrial processes is a prerequisite to harness their benefits in large-scale transformations for the synthesis of fine chemicals, food products, and pharmaceuticals. Immobilisation of wild-type or engineered enzymes using natural and synthetic carriers has been extensively employed to improve catalytic performance, ensure recovery and reuse, and thereby promote their use in industrial processes. Microgels as containers for protein immobilisation are advancing into a promising alternative as reflected by a growing body of literature that documents their use in sustainable catalytic processes. Microgels are porous, high molecular mass crosslinked submicron-sized colloidal polymer networks, which are swollen by the solvent in which they are dissolved (e.g., water). They can be designed to adjust their shape and volume upon external stimuli such as temperature, pH, ionic strength, and solvent nature. Microgels are ideal enzyme carriers due to their chemical and mechanical stability, tuneable architecture, biocompatibility, high water content, and their ability to achieve high enzyme loadings. In this review, we summarise the progress in the synthesis and applications of enzyme-loaded microgels (μ-Gelzymes). We start by exploring the different approaches used for enzyme immobilisation on or within microgels and give representative examples for the use of μ-Gelzymes in different applications. Subsequently, the potential of μ-Gelzymes in achieving sustainable catalysis is discussed from a green chemistry perspective. Finally, we draw future directions for further improvement of biocatalytic μ-Gelzymes as an emerging interdisciplinary research field of interactive soft matter.

  Overview of µ-Gelzymes categorised according to the methodologies for enzyme immobilisation Copyright: © Green Chemistry - Royal Society of Chemistry  

Figure Overview of µ-Gelzymes categorised according to the methodologies for enzyme immobilisation. Enzymes can be immobilised covalently on pre-synthesised microgels or immobilised during microgels synthesis. Alternatively, the stimuli-responsiveness of microgels (switchable enzyme carriers) can be utilised for enzyme immobilisation. Adapted from Nöth et al. (2020) with permission of the Royal Society of Chemistry.

 
 

Designed Streptococcus pyogenes Sortase A Accepts Branched Amines as Nucleophiles in Sortagging

Zhi Zou, Maximilian Nöth, Felix Jakob, and Ulrich Schwaneberg. Designed Streptococcus pyogenes Sortase A Accepts Branched Amines as Nucleophiles in Sortagging. Bioconjugate Chemistry, 2020

Sortase-mediated ligation (sortagging) is commonly performed using the Staphylococcus aureus sortase A (SaSrtA) that strictly recognizes the N-terminal glycine residue as sortase nucleophile. In this work, a rational design (site-directed mutagenesis close to the active site and β6/β7 loop engineering) of Streptococcus pyogenes sortase A (SpSrtA) for improved transpeptidase activity toward different N-terminal amino acid residues was conducted. The generated variant SpSrtA M3 (E189H/V206I/E215A) showed up to 6.6-fold (vs. SpSrtA wild-type) enhanced catalytic efficiency. Additionally, M3 retains the specificity toward N-terminal alanine, glycine, serine residues, as well as to branched primary amines (branched at the α-carbon) similar to the wild-type parent. Furthermore, M3 was applied for head-to-tail backbone cyclization of proteins.SpSrtA was engineered for increased activity towards differen N-terminal amino acid resiudes (alanine, glycine, and serine) and primary amines (branched at the α-carbon) by rational design. Copyright (2020), American Chemical Society.

  Sortase A Copyright: © Bioconjugate Chemistry
 
 

FhuA–Grubbs–Hoveyda Biohybrid Catalyst Embedded in a Polymer Film Enables Catalysis in Neat Substrates

Daniel Sauer and Tayebeh Mirzaei Garakani Copyright: © Bio VI

Tayebeh Mirzaei Garakani#, Daniel F. Sauer#, M.A. Stephanie Mertens, Jaroslav Lazar, Julia Gehrmann, Marcus Arlt, Johannes Schiffels, Uwe Schnakenberg, Jun Okuda, Ulrich Schwaneberg*, ACS Catalysis, 2020, 19, 10946-10953, DOI: 10.1021/acscatal.0c03055

Embedment of an artificial metalloprotein into a synthetic polymer film enables biohybrid catalysis in neat substrates.

Expanding synthetic capabilities by combining principles of biocatalysis (e.g., control of selectivity) with metal-catalyzed reactions that do not have their counterpart in nature is of high synthetic value. This manuscript reports for the first time biohybrid catalysis in neat substrates and not aqueous solutions. Being able to perform biohybrid catalysis in the “organic-solvent realm” expands the repertoire of metal catalysts that could be utilized to construct biohybrid catalysts opening exciting new opportunities in chemical synthesis. The biohybrid catalyst presented here consists of a Grubbs-Hoveyda type olefin metathesis catalyst embedded in the FhuA-protein, which is embedded in a poly (N-methyl pyrrole) matrix that stabilizes the FhuA-ß-barrel structure. Thereby, ring-closing metathesis in two different neat substrates has been performed. The here presented system has its strength especially in neat, hydrophobic substrates, where the biohybrid film performed 24-fold better compared to its homogeneous analogue. This work may inspire the catalysis community to regard organic solvent as suitable reaction media for biohybrid catalysis and explore the broad reaction scope of metal catalysis in engineered protein backbones for selective and synthetically valuable reactions.

This research was funded by the Bundesministerium für Bildung und Forschung (BMBF) in the framework of the BMBF-project “Chiral Membranes” (Förderkennzeichen: 031A164 and 031B0559) and the research award project “Hyka-synBio” (Förderkennzeichen: 031B0297). The authors further acknowledge the Deutsche Forschungsgemeinschaft (DFG) throug the international research training group “Selectivity in Chemo- and Biocatalysis” (IRTG 1628, SeleCa) for financial support. We thank Umicore, Frankfurt (Dr. A. Doppiu), for a generous gift of ruthenium precursor.

  Knight fights against fire-breathing dragon. Copyright: © Bio VI

Figure: A biohybrid catalyst consisting of the b-barrel protein FhuA equipped with a Grubbs-Hoveyda type catalyst as active site (sword) is embedded into a poly(N-methylpyrrole) matrix (knight’s armor), enabling the protein to withstand harsh conditions like neat substrates (dragon flame).

 
 

A photoclick‐based high‐throughput screening for the directed evolution of decarboxylase OleT

Ulrich Markel Copyright: © BIO VI

Ulrich Markel, Pia Lanvers, Daniel F. Sauer, Malte Wittwer, Gaurao V. Dhoke, Mehdi D. Davari, Johannes Schiffels, Ulrich Schwaneberg, Chemistry – A European Journal, 2020, DOI: 10.1002/chem.202003637

In this study, we present a photoclick assay that enables the high-throughput screening of decarboxylase variants in directed evolution.

Despite the synthetic importance of enzymatic oxidative decarboxylation, the directed evolution of decarboxylases is still hampered by the lack of high-throughput screening. In this study, we present a simple photoclick assay for the detection of styrenyl decarboxylation products. In a proof‐of‐principle study, the assay was applied for the directed evolution of the decarboxylase OleT. Our assay is compatible with both potential OleT operation modes, including the direct use of hydrogen peroxide as the enzyme’s co‐substrate or the use of a reductase partner protein. The screening of saturation mutagenesis libraries identified two enzyme variants that altered the substrate preference of the enzyme from long-chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to accelerate the directed evolution of OleT and other decarboxylases.

  A photoclick‐based high‐throughput screening for the directed evolution of decarboxylase OleT Copyright: © Chemistry – A European Journal Illustrative figure of the photoclick‐based high‐throughput screening for the directed evolution of a decarboxylase.
 
 

KnowVolution of a GH5 cellulase from Penicillium verruculosum to improve thermal stability for biomass degradation

Francisca Contreras Copyright: © BIO VI

Contreras, F., Thiele, M. J., Pramanik, S., Rozhkova, A. M., Dotsenko, A. S., Zorov, I. N., Sinitsyn, A. P., Davari, M. D. & Schwaneberg, U. ACS Sustainable Chemistry & Engineering. 2020, 8, 12388–12399. DOI: 10.1021/acssuschemeng.0c02465

Molecular knowledge on cellulases engineering for increased thermostability offers a high potential for sustainable biomass degradation.

Cellulases can be applied in lignocellulosic biomass degradation, feedstock, and pulp and paper production. In different applications, cellulases need to be active in high temperatures and harsh conditions. Cellulases are an expensive component in the processes; therefore, an increase in the cellulase lifetime will decrease the production costs and achieve more sustainable production. Nowadays, protein engineering has emerged as an important tool to improve enzyme properties. In this paper, we report a KnowVolution campaign of the cellulase PvCel5A from Penicillium verruculosum towards increased thermostability. The PvCel5A evolution shows that the KnowVolution engineering strategy, which combines experimental and computation work to minimize experimental efforts, was highly successful and generated a molecular understanding of the C-terminus’ role in improving thermal resistance. When compared to the wild type PvCel5A, variant PvCel5A-R17 presented an improvement of 5.5-fold in its half-life at 75 °C (from 32 to 175 min), and an increase in the melting temperature (Tm) by 7.7 °C (from 70.8 to 78.5 °C). Interestingly, the specific activity of PvCel5A was not reduced through the improvement of the melting temperature (high specific activity requires enzyme flexibility; thermal resistance requires strong interactions/rigid structures). Glycoside hydrolases from family 5 (GH 5) shares a (β/α)8-barrel, and the C-terminal region is a common feature within all enzymes in this family. Therefore, the stabilization concept can likely be transferred to other GH5 glycoside hydrolases. We believe the molecular knowledge of how to tailor cellulases for increased thermostability offers a great potential for enzymatic degradation and full exploitation of lignocellulosic biomass.

Computational biology division.

This work was funded from the Bundesministerium für Bildung und Forschung (BMBF) project EnzyBioDeg (FKZ: 031B0506) Bioeconomy international. MD simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under the project jara0187.

  KnowVolution protein engineering campaign towards improved thermostability of the cellulase PvCel5A. The specific activity of the variant R17 was improved in 100 % compared to wildtype at 75 °C. Copyright: © ACS Sustainable Chem. Eng. KnowVolution protein engineering campaign towards improved thermostability of the cellulase PvCel5A. The specific activity of the variant R17 was improved in 100 % compared to wildtype at 75 °C.
 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 
 

A colourimetric high-throughput screening system for directed evolution of prodigiosin ligase PigC

Stefanie Brands Copyright: © BioVI

Stefanie Brands, Hannah U. C. Brass, Andreas S. Klein, Jörg Pietruszka, Anna Joëlle Ruff, and Ulrich Schwaneberg, “A colourimetric high-throughput screening system for directed evolution of prodigiosin ligase PigC,” Chem. Commun., 2020, doi: 10.1039/D0CC02181D.

A two-step screening assay enables directed evolution of prodigiosin ligase PigC to perform biosynthesis of bioactive prodiginine compounds.

 
  Scheme of the PigC screening assay. Copyright: © Chem. Commun. Red and active colonies are preselected to be screened photometrically in 96-well format.

Prodigiosin synthetase PigC is a membrane bound ATP-ligase that catalyses the final condensation step in the biosynthesis pathway of the natural product prodigiosin, a tripyrrolic red pigment, which occurs naturally in several bacterial species. The compound class of prodiginines is famous for their prominent colour, which makes colonies of prodiginine-producing bacteria appear like little droplets of blood. Several prodiginine bioactivities have so far been discovered, which moved the compounds into the focus of industry: One artificial prodiginine derivative, Obatoclax Mesylate GX15-070, is currently on phase II clinical trials as candidate to treat different forms of cancer.

As chemical synthesis of prodiginines unfortunately is not trivial, biocatalysis remains a promising approach to get the compounds into hand. In our PigC screening assay, we combined chemical synthesis of prodiginine precursors (colourless in solution) with PigC biocatalysis (during which the red prodiginines are formed). In an agar prescreening, red and active colonies are selected for expression in 96-well format, where pyrrolic precursors are supplemented and prodiginines finally extracted and photometrically quantified by their red colour.

This concept makes the exchange of a pyrrolic precursor by a more promising pyrrole derivative easy, in our case, we screened with 2,3-dimethyl pyrrole instead of the natural PigC substrate 2-methyl-3-amyl pyrrole. Short-chain prodiginines have shown an increased bioactivity in anticancer autophagy tests. As 2,3-dimethyl pyrrole is not readily accepted by PigC, enzyme engineering campaigns on PigC are required to extend the synthetase’s substrate scope. This is where a suitable high-throughput screening method for detection of beneficial PigC variants comes into action: a PigC random mutagenesis library was generated in Pseudomonas putida KT2440 and screened for PigC variants with enhanced acceptance of the prodiginine precursor. After screening of approximately 3000 clones, a variant with one amino acid exchange in the substrate-binding domain stood out with a nearly 3-fold increased prodiginine yield. The variant’s catalytic efficiency with 2,3-dimethyl pyrrole (K M/k cat) proofed to be 2.9-fold higher over the wild type, and also its stability at 30°C was slightly increased, which enabled the production of higher amount of prodiginines in the screening assay.

This paper was written in the division Molecular Bioeconomy. The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Culture and Science within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-00213).

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Loop engineering of aryl sulfotransferase B for improving catalytic performance in regioselective sulfation

Yu Ji Copyright: © BioVI Yu Ji

Yu Ji, Shohana Islam, Haiyang Cui, Gaurao V. Dhoke, Mehdi D. Davari, Alan M. Mertens, Ulrich Schwaneberg, Catalysis Science & Technology, 2020, 10, 2369-2377 DOI: 10.1039/D0CY00063A

Reengineering of loop12 and loop 13 of aryl sulfotransferase B yielded variant with high sulfate transfer efficiency up to 94%.

  Method Copyright: © Catalysis Science & Technology Scheme/Figure 1 Overview of ASTB engineering strategy for improving ASTB’s catalytic performance.

Catechol sulfates act in our body as important antioxidants and often have anti-inflammatory properties. This study focuses on reengineering of loop 12 and loop 13 of aryl sulfotransferase B from Desulfitobacterium hafniense in order to improve the sulfate transfer efficiency of six catechols. The obtained aryl sulfotransferase B variants were generated in a KnowVolution campaign using the random mutagenesis method SeSaM and the multi-site saturation method OmniChange. The catalytic activity and catalytic efficiency of the final variant were improved for all six investigated catechols when compared to the wild type (e.g., 13.6-fold improvement of catalytic activity for 3-bromocatechol). HPLC-MS analysis confirmed the improved sulfate stoichiometry of aryl sulfotransferase B variant with a transfer efficiency up to 94% for 3-methylcatechol in comparison to 24% for the wild type. A molecular understanding of the improved sulfation activity of the variant was achieved through molecular docking studies and electron effects of catechol substituents were analyzed by Hammett equation. Enzymatic sulfation of catechol as well as substituted catechols by aryl sulfotransferases opens up an environmentally friendly route for chemo- and / or regioselective sulfation of valuable compounds, such as mycamine, sodium picosulfate, and minoxidil sulfate.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

In situ monitoring of membrane protein insertion into block copolymer vesicle membranes and their spreading via potential-assisted approach

Tayebeh Mirzaei Garakani (left) und Zhanzhi Liu (right) Copyright: © BioVI Tayebeh Mirzaei Garakani (left) und Zhanzhi Liu (right)

T. Mirzaei Garakani, Z. Liu, U. Glebe, J. Gehrmann, J. Lazar, M. A. S. Mertens, M. Möller, N. Hamzelui, L. Zhu, U. Schnakenberg, A. Böker, U. Schwaneberg, ACS Applied Materials & Interfaces, 2019, DOI: 10.1021/acsami.9b09302

A new approach to produce solid-supported biomimetic membranes via potential-assisted vesicle spreading was developed.

  Method Copyright: © ACS Applied Materials & Interfaces

Transmembrane proteins incorporated in solid-supported hybrid membranes can act as selective transporters or used for catalysis. The hydrophobic region of transmembrane proteins allows their incorporation into polymer membranes. The triblock copolymer poly(2-methyl oxazoline)-blockpoly(dimethylsiloxane)-block-poly(2-methyl oxazoline) and an engineered variant of the outer membrane protein Ferric hydroxamate uptake protein component A (FhuA) were used for the potential-assisted spreading of synthosomes on gold electrodes. FhuA lacking the cork domain (FhuA ∆1-160) was a useful candidate to achieve large passive diffusion channels in synthosomes. Isothermal titration calorimetry was used to analyze the insertion of FhuA into the polymer vesicles. The charge of the polymer was used to spread FhuA-containing vesicles on electrodes by electrostatic interactions. The potential-assisted vesicle spreading system was shown to produce homogenous synthosomes more efficiently than common spin- and dip-coating techniques. Functionality of the spread synthosomes was analyzed by passive ion transport response and through electrochemical impedance spectroscopy analysis. Solid-supported biomimetic membranes formed via potential-assisted spreading could be an interesting system for the development of new biosensors and as useful pores for drug delivery and water purification.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Engineering of laccase CueO for improved electron transfer in bioelectrocatalysis by semi-rational design

Dr. Lingling Zhang Copyright: © BioVI Dr. Lingling Zhang

Lingling Zhang, Haiyang Cui, Gaurao V. Dhoke, Zhi Zou, Daniel F. Sauer, Mehdi D. Davari, and Ulrich Schwaneberg, Chem. Eur. J., 2020. DOI: 10.1002/chem.201905598

The fifth copper binding site of CueO is very important to electron transfer kinetics of bioelectrocatalytic oxygen reduction.

  In silico 3D-Modell of CueO, zoomed into coordinated copper atom Copyright: © Chem. EUR. J. J. The structure of CueO (derived from PDB 3OD3) with the fifth copper coordinated by residues M355, D360, D439, and M441.

Different from other laccases, CueO is found to possess an extra copper binding site (i.e., the fifth copper binding site). To understand the role of the fifth copper in bioelectrocatalysis, semi-rational design was applied to generate a site-saturation mutagenesis library at the four coordination positions (M355, D360, D439, and M441). Thanks to the electrochemical screening platform established before ( Angew. Chem. 2019, 131, 4610 ), 11 improved variants were identified with over 2.5-fold increased currents. Molecular dynamics (MD) simulation suggested two reasons for the improvement: an increase in localized structural stability and a decrease of distance between the fifth copper and the first copper. It may guide a novel way to tailor laccases and perhaps other oxidoreductases for bioelectrocatalytic applications.

The simulation section was supported by division Computational Biology. This work was financed by Alexander von Humboldt Foundation and was immediately highlighted by Nature Reviews Chemistry once it was published.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Advances in ultrahigh-throughput screening for directed enzyme evolution

Ulrich Markel Copyright: © BioVI Ulrich Markel

Ulrich Markel, Khalil D. Essani, Volkan Besirlioglu, Johannes Schiffels, Wolfgang R. Streit and Ulrich Schwaneberg*, Chem. Soc. Rev., 2020, 49, 233-262. DOI: 10.1039/C8CS00981C

Here, we review state-of-the-art and up-and-coming ultrahigh-throughput methods for the screening of large libraries in directed enzyme evolution.

 
  Advances in ultrahigh-throughput screening for directed enzyme evolution Copyright: © Chemical Society Reviews State-of-the-art ultrahigh-throughput screening methods use cells and biomimetic compartments for the directed evolution of enzymes and are promising for the field of functional metagenomics.

Enzymes often need to be re-engineered or optimized in order to exploit their full potential. (Semi-) rational design requires detailed knowledge about structure function relationships. In turn, directed evolution methodologies can improve an enzyme’s properties without structural knowledge by iterative rounds of diversity generation and screening. Current diversity generation methods allow us to generate huge libraries but conventional screening on agar plates or in microtiter plates fails to interrogate the full generated diversity in reasonable time. Ultrahigh-throughput screening methods drastically increase the number of enzyme variants we can screen and speed up biocatalyst design ultimately widening our knowledge about sequence function relationships. In the present review, we summarize recent advances in ultrahigh-throughput screening for the directed evolution of enzymes. We discuss the importance of compartmentalization to link genotype and phenotype and illustrate how cells and biomimetic compartments can serve this function. Finally, we discuss how new functional metagenomics approaches can profit from ultrahigh-throughput screening to identify natural biocatalysts for novel chemical transformations.

 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

CompassR: A new rule for recombination of beneficial substitutions in directed evolution

Haiyang Cui Copyright: © BioVI Haiyang Cui

Haiyang Cui, Hao Cao, Haiying Cai, Karl Erich Jaeger, Mehdi D. Davari, Ulrich Schwaneberg, Chemistry A European Journal, 2019, https://doi.org/10.1002/chem.201903994

The Computer-assisted Recombination – CompassR – strategy is a selection filter for experimentalists to recombine beneficial substitutions in order to gradually improve enzyme performance and maximize improvements through recombination.

 
  CompassR works through the analysis of the relative free energy of folding and essentially acts as recombination pathfinder app for recombing beneficical substitutions that have been identified by directed evolution and/or computational design. Copyright: © Chem.: Eur. J. CompassR works through the analysis of the relative free energy of folding and essentially acts as recombination pathfinder app for recombing beneficical substitutions that have been identified by directed evolution and/or computational design.

Directed evolution has matured into a powerful method for improving enzymes in catalysis and medical applications as documented by the Nobel Prize in chemistry in 2018. A main remaining challenge 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. The latter limits researchers to harness the benefits from beneficial substitutions that have been identified in directed evolution campaigns and to exploit 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. The performance of CompassR was evaluated by analysis of 84 recombinants located on 13 positions of the Bacillus subtilis lipase A. Analysis of their relative free energy of folding was used to deduct the CompassR rule that substitutions with a relative free energy of folding below +0.36 kcal/mol can be efficiently recombined, variants with a free energy of folding between +0.36 and +7.52 kcal/mol were active and inactive indicating unpredictable behavior, folding energies over +7.52 kcal/mol mostly yielded inactive variants. The finally obtained recombinant with four amino acid substitutions had a 2.7-fold improved specific activity in 18.3 % ionic liquid [BMIM][Cl]. Notably, the BSLA variants with 3 and 4 substitutions were better performing than the variants with 2 substitutions. In essence, the deducted CompassR allows to recombine beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time-efficient manner.

This work was realized in the division Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0187). Haiyang Cui is financially supported by the China Scholarship Council (CSC) scholarship.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Chemoenzymatic cascade for stilbene production from cinnamic acid catalyzed by ferulic acid decarboxylase and an artificial metathease

Daniel Sauer, Stephanie Mertens Copyright: © BIO VI Daniel Sauer and Stephanie Mertens

M. A. Stephanie Mertens,‡ Daniel F. Sauer,‡ Ulrich Markel, Johannes Schiffels, Jun Okuda,* Ulrich Schwaneberg,* Catalysis Science & Technology, 2019, DOI: 10.1039/C9CY01412H

The combination of a decarboxylase and an artificial metathease in a chemoenzymatic cascade reaction for stilbene production with efficient removal of metal contamination is reported.

  Chemoenzymatic cascade for stilbene production from cinnamic acid catalyzed by ferulic acid decarboxylase and an artificial metathease Copyright: © Catalysis Science & Technology Combination of a decarboxylase and an artificial metathease in a chemoenzymatic cascade reaction for stilbene production

A chemoenzymatic cascade reaction involving a biocatalyst and a biohybrid catalyst for the production of stilbene derivatives was designed. Stepwise conversion of cinnamic acid as a renewable resource to valuable compounds was achieved in one pot in aqueous solution and under mild reaction conditions. The ferulic acid decarboxylase FDC1 from Saccharomyces cerevisiae was used for the conversion of cinnamic acid. In a following reaction, cross-metathesis of the styrene intermediate was performed with an artificial metathease, FhuA-GH, based on a Grubbs-Hoveyda catalyst incorporated into an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A, FhuA. Intermediate workup steps and isolation of the styrene intermediates was not required, as both reaction steps proceeded in aqueous solution. In comparison to the protein-free catalyst, cascade reactions with the artificial metathease revealed a significant lower metal content after a simple extraction step. The cascade reaction is the first example of the combination of biocatalysts and biohybrid catalysts for efficient removal of metal impurities in the product fraction.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Molecular understanding of enzyme and ionic liquid interactions provide a general protein engineering strategy for their sustainable biocatalytic applications

Dr. Gaurao Dhoke and Subrata Pramanik Copyright: © BIO VI Subrata Pramanik (left) and Dr. Gaurao Dhoke (right).

Pramanik S., Dhoke G. V., Jaeger KE., Schwaneberg U., Davari MD. ACS Sustainable Chem. Eng., 2019, https://doi.org/10.1021/acssuschemeng.9b00752 shared co-authorship

A general protein engineering strategy was suggested through molecular understanding of enzyme and ionic liquid interactions based on computational study.

  Comparative surface distribution of ionic liquid ions and water molecules on the surface of Bacillus subtilis lipase A. Copyright: © ACS Sustainable Chem. Eng. Comparative surface distribution of ionic liquid ions and water molecules on the surface of Bacillus subtilis lipase A.

In this publication, interactions between Bacillus subtilis lipase A (BSLA) and four imidazolium-based ionic liquids were studied using molecular dynamic simulations. This study provides first insight that ionic liquids co-solvents do not alter the overall and local BSLA conformation. The effect on the reduction of activity was attributed to surface interactions of the ionic liquid cations, which removed essential water molecules from the BSLA surface.Furthermore, the comparison of simulation results with experimental full site-saturation mutagenesis BSLA libraries confirmed that most of beneficial positions for resistance improvement are located at the ionic liquid cations binding regions. Taken together, these findings suggest that surface charge engineering might be a general protein engineering strategy to improve BSLA resistance in ionic liquids and is most likely applicable to other lipases and α/β-hydrolases.

This work was realized in the division of Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0187).

 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Science praises our recent research on plant pest management

Rainfast plant protection from disease by surface functionalizing dipeptides Copyright: © Green Chemistry Rainfast plant protection from disease by surface functionalizing dipeptides

The BiFuProt surface coating platform was highly appreciated in a recent news article published by Science as a new plant protection technology. The Science article quoted leading plant scientists. "With the current scale of the soybean rust problem, and the rapid evolution of resistance against multiple fungicides, any addition to the toolbox would be welcome," says Nichola Hawkins at Rothamsted Research in Harpenden, U.K. and Ralph Hückelhoven at the Technical University of Munich in Germany is quoted that the BiFuProt-technology is promising and "It opens a treasure box of solutions".

The protection of crops by pesticides is often challenging due to a very low persistence time and low rain fastness on the plant surface. A reduction of pesticide usage while preserving crop productivity demands new strategies for pest and disease control. In our current research highlight published in Green Chemistry we developed a platform technology that enables the functionalization of the aerial crop surface for sustainable disease management. We demonstrated an alternative way to protect soybean leaves from its most severe disease, the Asian soybean rust (Phakopsora pachyrhizi), by functionalizing the leaves with bifunctional fusion proteins or in short BiFuProts.

"With the current scale of the soybean rust problem, and the rapid evolution of resistance against multiple fungicides, any addition to the toolbox would be welcome."

- Nichola Hawkins (Rothamsted Research, Harpenden, Großbritannien)

The first domain binds to the wax layers of plant leaves, the second domain prevents the germination of Phakopsora pachyrhizi spores. In detail, the amphiphilic anchor peptides LCI, Thanatin, Tachystatin A2, and Lactoferricin B were genetically fused to the reporter protein eGFP and investigated for plant leaf binding. eGFP-LCI and eGFP-Thanatin strongly bound in a rainfast manner to the surface of soybean, barley, and corn leaves. Especially, eGFP-Thanatin bound to the soybean leaves and withstood high temperature, sunlight, and biotic degradation for at least 17 days. The weak binding of eGFP-Thanatin and eGFP-LCI to the wax-depleted mutant of barley or corn leaves indicated that the peptides mainly bind to the surface waxes of leaves. As a fusion partner for Thanatin, the antimicrobial peptide Dermaseptin 01 was selected. The bifunctional peptide Dermaseptin 01-Thanatin inhibited the germination of Phakopsora pachyrhizi spores in vitro and reduces Asian soybean rust disease by in a rainfast manner. It is very likely that state-of-the-art protein engineering strategies such as PePevo combined with a KnowVolution campaign will enable the design of peptides with tuned binding strength and persistence to match application demands. We expect that bifunctional peptides or proteins consisting of plant-attaching anchor peptides and pesticidal peptides or proteins have the potential to fight essentially any plant pest and disease in a rainfast manner.

This innovative research was performed in the framework of Bioeconomy Science Center (BioSC), which was funded by the Ministry of Culture and Science of the German State of North Rhine-Westphalia under the NRW Strategy Project BioSC. The BOOST FUND Project BiFuProts (Bifunctional fusion proteins for plant protection) team comprised Professor Ulrich Schwaneberg and Dr. Felix Jakob (initiator/coordinator; RWTH Aachen), Professor Uwe Conrath and Dr. Caspar Langenbach (RWTH Aachen University), Professor Lutz Schmitt (Heinrich-Heine-University Düsseldorf) and Professor Georg Noga, Dr. Mauricio Husche, and Dr. Shyam Pariyar (University of Bonn)

"It opens a treasure box of solutions".

- Ralph Hückelhoven (Technical University of Munich, Germany)

Plant health has already been an emerging research field within the Schwaneberg group in collaboration with plant scientist and chemists in last five year. The plant nutrient release technology (GreenGel) was successfully demonstrated on the example of cucumber iron deficiency in the BioSC funded project GreenGel. Dr. Felix Jakob and Professor Schwaneberg (coordinator; RWTH Aachen) were teamed up in GreenGel project with Professor Pich (RWTH Aachen University) and Professor Goldbach (University of Bonn). The GreenRelease technology is based on the compound loaded microgels (containers) that are decorated with plant adhesion promoting peptides and was published in Angewandte Chemie as hot paper in 2017. Based on the success of GreenGel and BiFuProts, the FocusLab greenRelease for Plant Health FocusLab has been funded (2.3 Mio€; for three years from January 2018 onwards) for translational research. The FocusLab is a part of BioSC as well and we teamed up with the groups of Professor Pich (RWTH Aachen University), Professor Conrath (RWTH Aachen University), Professor Noga (University of Bonn), Professor. Bröring (University of Bonn), Professor Knief (University of Bonn), Professor Groth (HHU Düsseldorf), Professor Gohlke (HHU Düsseldorf), and Professor Schurr (Forschungszentrum Jülich) to perform groundbreaking research in the field of plant health and protection in the upcoming years.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Tailor-made membrane channel enables chiral separation of racemates

Copyright: © BioVI Deepak Anand

Deepak Anand, Gaurao V. Dhoke, Julia Gehrmann, Tayebeh M. Garakani, Mehdi D. Davari, Marco Bocola, Leilei Zhu, and Ulrich Schwaneberg, Chemical Communications, 2019. DOI: 10.1039/c9cc00154a.

Chiral resolution of arginine enantiomers was achieved through an engineered Escherichia coli outer membrane protein FhuA.

  Schematic representation of chiral resolution of arginine racemate through engineered FhuA channel. Copyright: © Chem. Commun. Schematic representation of chiral resolution of arginine racemate through engineered FhuA channel.

Chiral molecules are of large economic value in chemical, pharmaceutical, and food industries. Separation of enantiomers can be achieved by various methods but it still remains a challenging task. Chiral protein-polymer membranes would be an attractive, cost-effective, and scalable alternative for chiral separation. The main challenges lie in the design of filter regions within the channel proteins and the development of screening systems to identify chiral channel protein variants. In the present study, we report for the first time a chiral β-barrel channel based on ferric hydroxamate uptake component A – FhuA, an outer membrane protein of E. coli. Two filter regions were identified and redesigned through screening of multi-site saturation mutagenesis libraries, in order to achieve the chiral separation of a D-/L-arginine racemate. Screening resulted in identification of FhuAF4 variant showing an improved enantiomeric excess of 24% at 52% conversion compared to the parent FhuA variant. Interestingly, even a subtle change of just two amino acids considerably influenced the selectivity of the FhuA channel. Steered molecular dynamic simulations indicated that the separation is based on diffusivity differences of two enantiomers through FhuAF4. It is likely that with the identified filter region and multi-site saturation libraries further improvements are achievable for separation of other amino acids and a broader range of enantiomers. The chiral FhuA channel proteins would be an excellent scaffold for generation of chiral membranes based on protein-polymer conjugates with a high potential for novel and scalable downstream processes.

We kindly acknowledge the German Federal Ministry of Education and Research (BMBF) for providing financial support to the Chiral Membranes I project. Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under the project RWTH0116.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Biohybrid catalysts for sequential one-pot reactions based on an engineered transmembrane protein

Dr. Daniel Sauer Copyright: © BioVI Dr. Daniel Sauer

D. F. Sauer, Y. Qu, M. A. S. Mertens, J. Schiffels, T. Polen, U. Schwaneberg*, J. Okuda*, Catal. Sci. Tech. 2019, 9, 942-946. DOI: 10.1039/C8CY02236D

Compartmentalization of organometallic catalysts was achieved by generation of their corresponding biohybrid catalysts based on the transmembrane protein FhuA to enable cascade reactions.

 
  Scheme of biohybrid catalysed one-pot reaction Copyright: © Catal. Sci. Tech. Synthesis of bibenzyl derivatives starting from styrene derivatives in a two-step one-pot fashion

In the present publication we showed that incorporation of organometallic catalysts into protein scaffolds enable cascade reactions in one pot, which were not possible by simply mixing the protein free catalysts. Bibenzyl derivatives (1,2-diphenylethane derivatives), which are valuable compounds in drug design or are used as building blocks in natural compound synthesis, were synthesized starting from styrene derivatives. In the first step, olefin cross-metathesis of the styrene derivatives was performed with a Ru-based Grubbs-Hoveyda type catalyst. In the subsequent step, hydrogenation of the stilbene intermediate was achieved with a Rh-type catalyst without isolation of the stilbene. This route was not possible by simply mixing the two organometallic catalysts in one pot. The strategy to compartmentalize the organometallic catalysts with proteins is promising approach and offers the possibility to keep reactions in a homogeneous fashion. The latter removes diffusion barriers through mass transfer between phases. This work was conducted in the division Next Generation Biocatalysis in cooperation with Professor Jun Okuda (Institute of Inorganic Chemistry, RWTH Aachen University). This research was financially supported by the DFG and the BMBF. A generous gift of metal precursors by Umicore, Frankfurt, is acknowledged.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 
  Illustration of methods and results of the publication Copyright: © Angew. Chem. Int. Ed Schematic illustration of the electrochemical screening platform, structural view of the copper and the adjacent residues of CueO and electrocatalytic polarization curves with respect to CueO wildtype and CueO D439T/L502K.

In this study, the bacterial laccase CueO from Escherichia coli was chosen to perform directed evolution towards lowering the overpotential of cathodic oxygen reduction. A robust and efficient 8-channel electrochemical screening platform was developed for the first time to evaluate CueO variants generated by random mutagenesis and subsequent site-saturation mutagenesis (SSM). The enzyme immobilization could be accomplished in 20 seconds and directly from crude cell lysates. Two positions adjacent to the coordinated ligands of the T1 copper site, was identified as a main region that contribute to improvements in the onset potential. A remarkable increased onset potential of 0.54 V was obtained with two amino acid substitutions. Finally, the cathode generated open circuit potential of 0.56 V as well as 1.72-fold enhanced power output for the enzymatic biofuel cell comprising the generated CueO variant coupled with a glucose dehydrogenase. The developed directed evolution protocol offers a promising methodology beyond chemistry and materials science in improving laccase properties such as substrate specificity, turnover efficiency, and tolerance to harsh-environment conditions.

This work was supported by the Alexander von Humboldt Foundation.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Targeting microplastic particles in the void of diluted suspensions

Photos of Shohana Islam and Lina Apitius Copyright: © BioVI

Shohana Islam#, Lina Apitius#, Felix Jakob, and Ulrich Schwaneberg. 2019, Environ. Int. 123: 428-435. #shared co-authorship. DOI.org/10.1016/j.envint.2018.12.029

In the present publication, the application of the anchor peptide Tachystatin A2 as adhesion promoter is described, which enhances the degradation of polyester-polyurethane nanoparticles by the cutinase Tcur1278.

 
 

Directed evolution of bacterial laccase CueO for enzymatic biofuel cells

Dr. Lingling Zhang Copyright: © BioVI

Lingling Zhang, Haiyang Cui, Zhi Zou, Tayebeh Mirzaei Garakani, Catalina Novoa-Henriquez, Bahareh Jooyeh, and Ulrich Schwaneberg, Angew. Chem. Int. Ed, 2019. DOI: 1002/ange.201814069

Directed evolution of CueO via electrochemical screening found substitutions that led to the dramatic overpotential decrease of 0.12 V, making CueO competitive to fungal laccases and applicable in enzymatic biofuel cells.

  Field emission scanning electron microscopic image of microplastic degradation by cutinases Copyright: © Environ. Int. Field-scanning electron micrograph of the enzymatic degradation of polyester-polyurethane after 24 h. Only when treated with the cutinase anchor fusion protein is a degradation of the material to visible..

The accumulation of microplastics in the environment as well as in the food chain will be a grand upcoming challenge for our society. Polyurethanes belong to the accumulating plastics as they are widely used in medical, e.g. catheters, and industrial products, especially as foams. Polyurethane is a man-made polymer and therefore not abundant in nature. Hence, only a few fungal and microbial strains as well as enzymes, such as polyurethaneases and cutinases, have been reported to efficiently degrade polyurethane. An estimated degradation of plastic in nature takes at least 50 to 100 years. A minimization of existing environmental pollution requires sustainable management strategies for microplastics. Material binding peptides like anchor peptides strongly bind to synthetic polymers such as polypropylene, polyethylene terephthalate, and polyurethane. In this publication, we report the fusion of the anchor peptide Tachystatin A2 to the bacterial cutinase Tcur1278. The fusion enzyme Tcur1278-Tachystatin A2 accelerated the degradation of polyester-polyurethane nanoparticles by a factor of 6.6 in comparison to wild-type Tcur1278 without any anchor peptide. Additionally, the degradation half-life of polyester-polyurethane nanoparticles was reduced from 41.8 h to 6.2 h indicating a 6.7-fold improvement in a highly diluted polyester-polyurethane suspension by the fusion enzyme. Taken together, anchor peptides provide a versatile tool for a targeted degradation of micro- and nanoplastics at ambient temperature in highly diluted particle suspensions.

This work was financed by the German Federal Ministry of Education and Research and the German Federation of Industrial Research Associations. We acknowledge Prof. Wolfgang Zimmermann and Dr. Ren Wei for the sequence of the cutinase Tcur1278. We thank Thorsten Palmer for dynamic light scattering measurements and finally the Center for Chemical Polymer Technology especially Sabrina Mallmann for field emission scanning electron microscopy.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Towards evolution of artificial metalloenzymes – A protein engineer’s perspective

Photos of Ulrich Markel and Daniel Sauer Copyright: © BioVI

Ulrich Markel,# Daniel F. Sauer,# Johannes Schiffels, Jun Okuda, and Ulrich Schwaneberg, Ang. Chem. Int. Ed., 2018 , DOI: 10.1002/ange.201811042

In the present publication, an overview of the early approaches of directed evolution of biohybrid catalyst is given.

 
  Scheme of directed evolution of biohybrid catalysts Copyright: © Ang. Chem. Directed evolution of artificial metalloenzymes/biohybrid catalysts. In contrast to the directed evolution of natural enzymes, the directed evolution of artificial metalloenzymes/biohybrid catalysts requires two additional steps (orange).

The incorporation of artificial metal-cofactors into protein scaffolds yields a new class of catalysts termed biohybrid catalysts or artificial metalloenzymes. In addition to modification of the artificial cofactor, these biohybrid catalysts can be modified at the second coordination sphere provided by the protein scaffold. Protein engineering provides tremendous potential to tailor such biohybrid catalysts but requires a robust screening system and sophisticated metal cofactor conjugation. In this minireview, we give an overview of the recent efforts in this field. We describe high-throughput screening methods applied for the directed evolution of biohybrid catalysts and we illustrate how non-chiral catalysts catalyze reactions enantioselectively by highlighting the first successes in this emerging field. Furthermore, we summarize the potential and limitations that need to be overcome to advance from biohybrid catalysts to true artificial metalloenzymes.

We gratefully acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) through the International Research Training Group ‘Selectivity in Chemo- and Biocatalysis’ (SeleCa) and the Bundesministerium für Bildung und Forschung (BMBF) (FKZ: 031B0297). #: These authors contributed equally

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Enzyme-polyelectrolyte complexes boost the catalytic performance of enzymes

Photo of Martin Thiele and Mehdi D. Davari Copyright: © Bio VI

Martin J. Thiele, Mehdi D. Davari, Melanie König, Isabell Hofmann, Niklas O. Junker, Tayebeh Mirzaei Garakani, Ljubica Vojcic, Jörg Fitter, Ulrich Schwaneberg, ACS Catalysis, 2018, DOI: 10.1021/acscatal.8b02935

In the present publication, a molecular understanding was reported on polyelectrolyte-enzyme complexes which boost enzymatic activity in multi-component systems.

 
  Schematic of the boosting effect of polyelectrolytes on proteases Copyright: © ACS Boosting the catalytic performance of protease 1ST3 through the interaction of polyelectrolytes on surface amino acid residues close to the Ca2+ binding site of the protease.

In this publication, a molecular understanding of the boosting effect of polyacrylic acid -PAA-) and poly-L-γ-glutamic acid -γ-PGA- for a nonspecific subtilisin protease was generated through biophysical characterization, e.g. fluorescence correlation and circular dichroism spectroscopies, isothermal titration calorimetry, molecular dynamics simulations and protease reengineering, e.g. site-saturation mutagenesis. This study revealed that enthalpically driven interactions via key amino acid residues close to the protease Ca2+ binding sites cause the boosting effect in protease activity. On the molecular level electrostatic interactions result in the formation of protease-polyelectrolyte complexes. Site-saturation mutagenesis on 6 positions yielded an increased proteolytic performance against a complex protein mixture -trademark CO3; up to ~300% and ~70%- in the presence of PAA and γ-PGA. Being able to fine-tune interactions between proteins and negatively charged polymers through integrative use of computational design, protein reengineering and biophysical characterization proved to be an efficient workflow to improve protease performance.

This research was partially funded by Henkel AG & Co. KGaA, Düsseldorf, Germany, as part of the Henkel Innovation Campus for Advanced Sustainable Technologies -HICAST- project. Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under projects RWTH0116 and JARA0169.

 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

Directed evolution of sortase A for efficient site-specific bioconjugations in organic co-solvents

Photo of Zhi Zou Copyright: © Bio VI

Evolved sortase A variants enable efficient site-specific conjugation of peptide/amine in organic co-solvents

Directed evolution of sortase A in 45% (v/v) DMSO, as co-solvent, yielded variants M1 and M3 with 2.2-fold increased resistance and 6.3-fold increased catalytic efficiency compared to sortase A wild-type, respectively. The generated variants were used to expand sortase-mediated ligation in organic co-solvent with hydrophobic substrates. Variant M3 showed up to 4.7-fold increased activity for peptide-peptide/peptide-amines in DMSO/DMF co-solvents compared to the wild-type. Structure-function relationship of sortase A in DMSO co-solvent were investigated by computational studies, which revealed that conformational mobility is important for the resistance gained by sortase A in organic co-solvent.

 
  Scheme Directed Evolution of Sortase A Copyright: © Royal Society of Chemistry Schematic representation of sortase A -M3 mediated site-specific ligation in organic co-solvent

This work was financed by the Chinese Scholarship Council-CSC. Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under projects RWTH0116 and JARA0169.

For more detail, access this publication in our Research Highlights and Chemical Communications website.

 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 

KnowVolution campaign of an aryl sulfotransferase increases activity toward cellobiose sulfatation

Photo of Shohana Islam Copyright: © Bio VI Shohana Islam, first author of the study on a KnowVolution campaign of an aryl sulfotransferase toward cellobiose sufatation

Islam, S., Laaf, D., Infanzón, B., Pelantová, H., Davari, M. D., Jakob, F., Křen, V., Elling, L., and Schwaneberg, U. Chemistry - A European Journal , 2018 . doi:10.1002/chem.201803729

Glycosaminoglycans are mostly sulfated polysaccharides playing significant roles in signal transduction, anti-coagulation, detoxification, and many more. Full chemical synthesis of glycosaminoglycans is still a very hard task due to many synthesis steps that affect the final yields. A suitable and sustainable alternative is represented by the in vitro synthesis of sulfated polysaccharides such as cellulose or chitin that mimic functionalities of glycosaminoglycans. Enzymatic sulfation of polysaccharide building blocks by sulfotransferases is synthetically attractive due to their ability to perform highly chemoselective sulfation in aqueous solution and at ambient temperature. The bacterial aryl sulfotransferase B - ASTB was reengineered to improve the sulfation activity toward the cellulose building block, cellobiose. A full KnowVolution campaign was performed. After screening 3,067 ASTB variants, Leu446 and Val579 were identified as beneficial positions to show positive effects on the ASTB activity.

 
Scheme for sulfation of sugar and glycosaminoglycans Copyright: © Bio VI Figure: ASTB was reengineered by KnowVolution toward a versatile tool for sulfation of sugar and glycosaminoglycans.

Advancement of an aryl sulfotransferase toward a synthetically attractive sulfation agent for saccharides

Computational studies suggested that Leu446Pro conveys more flexibility to the substrate-binding site and Val579Lys is a distal substitution. Finally, the recombination of Leu446Pro and Val579Lys yielded a variant ASTB-M5 with up to 7.6-fold increased specific activity of ASTB toward cellobiose. A monosulfation of cellobiose was confirmed via mass spectrometry indicating a very selective sulfation. Furthermore, conversion was increased from 33.8% to 87.1% by the ASTB variant in the synthesis of the monosulfated glycosaminoglycan-building block, N-acetylglucosamine. Structure elucidation confirmed a partially regioselective monosulfation at C-3 and C-4 position, opposed to the chemically preferred C-6.

This work was a successful collaboration between Professor Schwaneberg and Professor Elling from RWTH Aachen and Professor Křen from Czech Academy of Sciences. Our work was financed by the projects FuPol, BioSulfa and the German Federal Ministry of Education and Research. For more detail, access this publication in our Research Highlights and Chemistry – A European Journal website.

 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 
 

Directed OmniChange evolution converts P450 BM3 into an alkyltrimethylammonium hydroxylase

Photo of Yu Ji Copyright: © Bio VI Yu Ji, first author of the study on Omnichange evolution of P450BM3 into a alkyltrimethylammonium hydroxylase

Yu Ji, Alan Maurice Mertens, Christoph Gertler, Sallama Fekiri, Merve Keser, Daniel F. Sauer, Kilian E. C. Smith, and Ulrich Schwaneberg*, Chemistry - A European Journal, 2018, doi:10.1002/chem. 201803806

 
 

Reengineering of the P450 BM3 substrate specificity towards the hydroxylation of CTAB by OmniChange multi-site mutagenesis method

 
  Image of reaction educts and products Copyright: © Chemistry – A European Journal Directed OmniChange evolution converts P450 BM3 into an alkyltrimethylammonium hydroxylase.

Bolaform surfactants are a novel class of compounds with a wide range of industrial and technical applications. Bolaform surfactants are capable of forming of very small micelles and therefore are more effective than contemporary surfactants.

However, their production is expensive and involves the use of strong acids and large amounts of solvents. An alternative green synthesis route is the direct hydroxylation through monooxygenases as performed in nature.

 
 

In this study, the OmniChange multi-site mutagenesis method was applied for reengineering of the P450 BM3 substrate specificity towards the hydroxylation of CTAB by simultaneous mutagenesis of four relevant positions. Improved variants were identified in a two-step screening system. Then 10 promising P450 BM3 variants were analyzed by HPLC-MS/MS. Four P450 BM3 variants had significantly improved productivities and were kinetically characterized after purification. Interestingly all four variants were capable to di-hydroxylate CTAB and coupling efficiency up to 92.5% were obtained.

Notably, di-hydroxylation products of CTAB with bolaform surfactant properties have for the first time been produced. Bolaform surfactants are biodegradable and sustainable surfactants that offer excellent solubility properties and novel possibilities for drug delivery and/or compound formulations. Additionally, the two-step screening system proved to be efficient to boost coupling efficiency and can likely be used in many other P450 evolution campaigns to generate robust P450 catalysts. This work was funded by the China Scholarship Council, No. 201608080082.

Please find the link to this publication here.

 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 
 

Enzyme-compatible dynamic nanoreactors from electrostatically bridged like-charged surfactants and polyelectrolytes

Photo of M. Thiele and M.D.Davari

Martin J. Thiele, Mehdi D. Davari, Isabell Hofmann, Melanie König, Carlos G. Lopez, Ljubica Vojcic, Walter Richtering, Ulrich Schwaneberg and Larisa A. Tsarkova, Angewandte Chemie, 2018, DOI: 10.1002/anie.201805021

 
 

In the present publication, a discovery of nanoreactors from electrostatically bridged like-charged surfactants and polyelectrolytes was reported. Incorporation of a protease into such dynamic nanoreactors results in a synergistically enhanced cleaning performance due to the improved solubilization accessibility for the protease through the interaction with the nanoreactors.

 
 

An unanticipated mechanism of attractive electrostatic interactions of fully neutralized polyacrylic acid with like-charged surfactants is reported. Amphiphilic polymer-surfactant complexes with high interfacial activity and a solubilization capacity exceeding that of conventional micelles are formed by bridging with Ca2+ ions. Incorporation of a non-specific protease into such dynamic nanoreactors results in a synergistically enhanced cleaning performance due to improved solubilization of poorly water-soluble immobilized proteins. Competitive interfacial and intermolecular interactions on different time- and length-scales have been resolved using colorimetric analysis, dynamic tensiometry, light scattering, and molecular dynamic simulations. The discovered bridging association mechanism suggests reengineering of surfactant/polymer/enzyme formulations of modern detergents and opens new opportunities in advancing labile delivery systems.

 
  Schematic presentation of the nanoreactor´s mode of action Copyright: © Bio VI

Image: Scheme of an unanticipated mechanism of attractive electrostatic interactions of fully neutralized polyacrylic acid (PAA) with like-charged surfactant SLES. Amphiphilic polymer-surfactant complexes with high interfacial activity and solubilization capacity are formed by bridging with Ca2+ ions. Incorporation of a protease into such dynamic nanoreactors results in an enhanced cleaning performance because of the improved solubilization of poorly water-soluble immobilized proteins.

 
 

This research was partially funded by Henkel AG & Co. KGaA, Düsseldorf, Germany, as part of the Henkel Innovation Campus for Advanced Sustainable Technologies (HICAST). Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under projects RWTH0116 and JARA0169. L.A.T. acknowledges financial support by the Russian Foundation for Basic Research (RFBR) according to the research project No 18-53-76005.

 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 
 

A loop engineering strategy improves laccase lcc2 activity in ionic liquid and aqueous solution

Photo of Anne Wallraf Copyright: © Bio VI

A. M. Wallraf, H. Liu, L. Zhu, G. Khalfallaha, C. Simons, H. Alibiglou, M. D. Davari and U. Schwaneberg, Green Chemistry, 2018, DOI: 10.1039/C7GC03776G

 
 

Importance of a domain-connecting loop in laccase for increased activity in ionic liquid (IL) was identified.

Laccases are involved in lignin degradation. EMIM- and BMIM-based ionic liquids -IL- show excellent solubilization of wooden biomass but impede laccase activity. Protein engineering to improve the activity and resistance of laccases in ILs is promising for lignin valorization for the sustainable production of fuels and bulk high-value chemicals . We report for the first time an efficient semi-rational design with focus on a domain-connecting loop of a laccase lcc2 (loop L1) from Trametes versicolor.

The loop engineering strategy is based on a KnowVolution campaign and can be divided into three steps. Prediction of seven resistance increasing positions out of 37 amino acids in L1 - residues 284-320 - was performed by in silico SSM analysis with FoldX. These seven positions were saturated by SSM and four beneficial positions were subjected to simultaneous SSM using OmniChange. The OmniChange variants OM1 -A285P/A310R/A312E/A318G- and OM3 -A310D/A312P/A318R- showed a 3.9-fold -535.8 ± 36.9 U/mg- and 1.6-fold -216.8 ± 5.3 U/mg increased specific activity in aqueous solution -lcc2 WT, 138.9 ± 6.5 U/mg-, respectively, and up to 8.4-fold increased activity in 35% EMIM EtSO4 and aqueous solution when compared to lcc2 WT. Hydrogen bond pattern analysis revealed that both variants harbor an increased number of hydrogen bonds within the loop and between domains two and three which resulted in increased IL resistance.

 
  Flow scheme of the loop engineering experiment Copyright: © Royal Society of Chemistry
 
 

Entropy analysis indicated that the substitution of alanine at each selected amino acid position A285, A310, A312, and A318 reduced the flexibility of the loop L1. Conservational analysis with ConSurf server showed that the long domain-connecting loop L1 is a conserved feature in fungal laccases and suggests loop engineering as a useful strategy for increasing laccase activity in ILs and aqueous solutions. This work was funded by Deutsche Forschungsgemeinschaft DFG in the frame of the research cluster “ Tailor-Made Fuels from Biomass ” TMFB.

 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 
 

Cavity size engineering of a beta-barrel protein generates efficient biohybrid catalysts for olefin metathesis

Photo of Alexander Grimm and Daniel Sauer Copyright: © Bio VI

Grimm, A.R.*, Sauer, D.F.*, Davari, M.D., Zhu, L., Bocola, M., Kato, S., Onoda, A., Hayashi, T., Okuda, J., Schwaneberg, U.; ACS Catalysis, 2018, 8, pp 3358–3364, DOI: 10.1021/acscatal.7b03652. (*contributed equally)

 

The successful application of the presented cavity size engineering strategy to biohybrid catalysis can potentially be transferred to other beta-barrel protein scaffolds to generate cavity sizes that match the sterical demands of synthetic catalysts.

 
  biohybrid catalyst Copyright: © Bio VI

By incorporating a synthetic metal catalyst into a protein scaffold, a biohybrid catalyst is obtained. The protein scaffold can potentially alter the selectivity of the metal catalyst.and solubilizes it in water, while retaining their broad reaction scope. What’s new about this work is that until now, protein scaffolds for synthetic catalysts have mainly been engineered by exchanging individual amino acids. While a lot has been achieved using this conventional strategy, it is limited by the number of amino acids in the protein available for substitution. Here, cavity size engineering of a beta-barrel protein called nitrobindin was performed by duplicating multiple beta-strands to generate an expanded variant.

 
 

"Incorporation of a synthetic metal catalyst into a protein scaffold yielded a biohybrid catalyst that combines remarkable performance in aqueous media with the broad reaction scope of organometallic catalysts."

Alexander R. Grimm

It is the first time this has been done for biohybrid catalysis. By duplicating entire beta-strands, it was possible to covalently incorporate bulky catalysts and achieve excellent conversions in a whole series of olefin metathesis reactions – carbon-carbon double bond formation reactions. What’s exciting about this is that the design strategy that was used can potentially be transferred to other beta-barrel protein scaffolds to generate cavity sizes that match the sterical demands of synthetic catalysts. If high-throughput screening is applied to these new variants in the future, it should be possible to explore directed biohybrid catalyst evolution much more efficiently, which will likely further increase performance. This work was made possible through funding from the Deutsche Forschungsgemeinschaft and Bundesministerium für Bildung und Forschung.

Read more

 
  Biohybrid Catalyst Copyright: © ACS Catalysis Figure 1 Comparison of conversion of cavity size engineered biohybrid catalyst and protein-free catalyst in ring opening metathesis polymerization.
 
 
 
 

A Whole Cell E. coli Display Platform for Artificial Metalloenzymes: Polyphenylacetylene Production with a Rhodium-Nitrobindin Metalloprotein

Photo of Alexander Grimm Copyright: © Bio VI

Grimm, A.R., Sauer, D.F., Polen, T., Zhu, L., Hayashi T., Okuda J., Schwaneberg, U. (2018). A whole cell E. coli display platform for artificial metalloenzymes: polyphenylacetylene production with a rhodium-nitrobindin metalloprotein. ACS Catal., 8, 2611-2613.

 
 

A Whole Cell E. coli Display Platform for Artificial Metalloenzymes

 
  Function principle of the whole cell catalyst Copyright: © Bio VI

In the latest publication of our colleageus from the Hybrid Catalysis & High Throughput Screening division, the first bacterial cell surface display-based whole cell biohybrid catalyst, termed ArMt bugs, was generated, characterized and applied to the stereoselective polymerization of phenylacetylene. Whole cell catalysis is very important for the cost-effective production of chemicals by biotechnological means. Despite the promising application of whole cells to biohybrid catalysis, researchers worldwide had to face the inactivation of their valuable synthetic metal catalysts within cells due to abundant thiols.

 
 

The authors therefore armed the surface of their Escherichia coli whole cells with rhodium-nitrobindin biohybrid catalysts via an esterase-based autotransporter to separate the catalysts from inhibiting cellular compounds. A high turnover number of 39,000,000 per cell in the polymerization of phenylacetylene and potential applications in directed evolution and high-throughput screening make the ArMt bugs an attractive platform for bioorthogonal reactions. This work was made possible through funding from the Deutsche Forschungsgemeinschaft and Bundesministerium für Bildung und Forschung.

Read more

 
  Functional principle whole cell catalyst Copyright: © Bio VI
 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 
 

An enzymatic route to α-tocopherol synthons: Aromatic hydroxylation of pseudocumene and mesitylene with P450 BM3

A.Denning/A.Weingartner Copyright: © Bio VI

Dennig*, A., Weingartner*, A. M., Kardashliev, T., Müller, C. A., Tassano, E., Schürmann, M., Ruff, A. J., Schwaneberg, U. (2017). Chemistry - European Journal , first published online: October 9 2017, DOI: 10.1002/chem.201703647

 
 

P450 BM3 variants for the production of key phenolic building blocks for α-tocopherol synthesis

 
  Hydroxylation through P450BM3 Copyright: © Wiley VCH

Alternative enzymatic routes for vitamin E synthesis are important to match increasing demands. Here, we show the first direct aromatic hydroxylation of pseudocumene and mesitylene in water, with O2 and under mild reaction conditions to access five key phenolic α -tocopherol synthons including a direct route to trimethylhydroquinone, TMHQ. The P450 BM3 wild-type catalyzed a 94% selective aromatic hydroxylation of mesitylene, whereas pseudocumene was hydroxylated to a large extent ranging from 46-64% on benzylic positions. Site-saturation mutagenesis generated a new P450 BM3 mutant, named “variant M3”, with a 3 to 8fold increased coupling efficiency and 75 to 230-fold increased activity for pseudocumene and mesitylene conversion. Additional π - π interactions introduced by mutation A330F improved not only productivity and coupling efficiency, but also a 61 to 75% selectivity toward aromatic hydroxylation of pseudocumene. Detailed product pattern analysis, substrate docking and mechanistic considerations support the hypothesis that pseudocumene binds in an inverted orientation in the active site of P450 BM3 WT as compared to P450 BM3 variant M3 to allow this change in chemo-selectivity.

Under continuous NADPH-recycling the novel P450 BM3 variant M3 was able to produce the key tocopherol precursor TMHQ at 35% selectivity at concentrations up to 0.18 mg ml-1 directly from pseudocumene, which is a significant step toward more sustainable synthesis of vitamin E. The reaction can be performed in one-pot without the need for intermediate purification or additional catalysts. In case of mesitylene over-oxidation leads to dearomatization and formation of a valuable p-quinol synthon that can directly serve as educt for synthesis of TMHQ. This study provides an enzymatic route to key phenolic synthons for α-tocopherols and first catalytic and mechanistic insights into direct aromatic hydroxylation and dearomatization of trimethylbenzenes with O2.

Read more

 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 
 

Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3-Hydroxyalkanoates

Yunus Ensari & Gaurao Dhoke Copyright: © Bio VI

Yunus Ensari, Gaurao V. Dhoke, Mehdi D. Davari, Marco Bocola, Anna Joëlle Ruff, Ulrich Schwaneberg, Chemistry – A European Journal 2017, 23, 51, Issue, 12636-12645.

 
 

Enlargment of substrate binding pocket of cpADH5 led the conversion of medium chain methyl 3-hydroxyalkanoates and inverted enantiopreference for short chain methyl 3-hydroxyalkanoates.

 
  cpADH5 Copyright: © Chemistry – A European Journal Scheme 1: General overview of cpADH5 binding pocket engineering.

Selective oxidation of primary or secondary alcohols to carbonyl compounds, i.e. aldehydes and ketones, is a key reaction in organic synthesis and industry. Oxidation of alcohol to ketones can be performed efficiently by a great number of chemical oxidation methods. Selectivity, undesired by-product formation, substrate scope and conversion rates are still major challenges in oxidation of alcohols. Alcohol dehydrogenases are promising alternatives to metal catalysts and perform reversible oxidations of primary or secondary alcohols to their corresponding aldehydes or ketones.

We hope that the enzymatic oxidation of 3-hydroxy-FAMEs will open novel synthetic routes to attractive building block in the synthesis of pheromones and b-lactam antibiotics.

Gaurao V. Dhoke

Ketone-functionalized fatty acid derivatives are versatile compounds in organic chemistry, are used as intermediates and building blocks in the production of pharmaceutically active compounds, detergents, and so on.

This study reports the first reengineered Candida parapsilosis alcohol dehydrogenase - cpADH5 - for the efficient oxidation of methyl 3-hydroxyhexanoate and methyl 3-hydroxyoctanoate, which were not converted by the cpADH5 WT, to their corresponding ketones. In this study, we mutated and redesigned the substrate binding pocket of cpADH5 in order to extend its substrate scope toward medium-chain 3-hydroxy fatty acid methyl esters – FAME- which contain 6--12 carbon atoms in their alkyl chains. Furthermore, we describe the inverted enantiopreference for the oxidation of methyl 3-hydroxybutyrate which was observed for our engineered cpADH5 variant.

Read more

 
 

------------------------------------------------------------------------------------------------------------------------------------------------------

 
 

Research highlight: Micro delivery service for fertilizers

Micro delivery service for fertilizers Copyright: © Wiley-VCH

Plants can absorb nutrients through their leaves as well as their roots. However, foliar fertilization over an extended period is difficult. In the journal Angewandte Chemie, German researchers have now introduced an efficient delivery system for micronutrients based on biohybrid microgels. Special peptides anchor the "microcontainers" onto the leaf surface while binding sites inside ensure gradual release of the "cargo".

Foliar fertilization is already commonly used in areas such as viniculture, when the leaves on the vines turn yellow due to a mineral deficiency. Yet, despite the use of detergents, adhesives, and humectants, controlled delivery of nutrients through foliar fertilization over several weeks is nearly impossible to achieve. Up to 80% of the nutrients are washed away, winding up in the soil and being converted into forms that the plant cannot use. In addition, they can be washed into bodies of water and cause environmental problems. An additional problem is that strong sunlight evaporates the water out of the applied fertilizer solution. This results in a high salt concentration that draws water out of the leaf and causes burn damage.

A team from DWI-Leibniz Institute for Interactive Materials in Aachen, RWTH Aachen University, and the University of Bonn has now developed a foliar fertilization system based on biocompatible microgels that adhere selectively to leaves for a long period and slowly deliver nutrients in a controlled fashion. Microgels are tiny particles of cross-linked macromolecules that can bind water and other molecules, such as fertilizers very efficiently.

Led by Ulrich Schwaneberg, Andrij Pich and Felix Jakob, the researchers equipped the interiors of gel particles with binding sites modeled on the iron-binding proteins of bacteria. These ensure that the iron ions are released slowly. The microgels are loaded with an iron-containing solution at a pH of 3. When the pH rises to 7, the microgels shrink, releasing water and binding the iron.

The surface of the gel particles is equipped with anchor peptides from lactic acid bacteria. These bind securely to leaf surfaces to hinder rinsing away of the microgels. The water in the gel provides an aqueous microenvironment that allows the iron to diffuse into the leaves. Yellow leaves of iron-deficient cucumber plants rapidly turned green in spots where the new foliar fertilizer was applied.

By incorporating different binding sites, the microgel "containers" can be loaded with a multitude of other metal ions or agents. A controlled delivery of agents as required would minimize the applied quantities as well as the release of fertilizers and pesticides into the environment. Low production costs, high levels of loading, easy application, and adjustable adhesive properties should make broad industrial applications possible. The goal is to make self-regulating delivery systems for sustainable agriculture.

Richard A. Meurer et al, Biofunctional Microgel-Based Fertilizers for Controlled Foliar Delivery of Nutrients to Plants, Angewandte Chemie International Edition (2017). DOI: 10.1002/anie.201701620

Read more

 
 

Generation of building blocks for synthesis of filter membranes

Generation of building blocks for synthesis of filter membranes Copyright: © Bio VI

The iron transporter FhuA - Ferric hydroxamate uptake protein component A is a channel protein produced by the bacterium Escherichia coli which was tailored for the generation of synthetic membranes using FhuA proteins and polymers. The FhuA protein forms a permeable channel with a uniform pore size from 2.5 to 3.0 nm and a barrel-like structure. Lysine residues were specifically located in a rim on the outer surface of the channel above the transmembrane region for the attachment of polymer chains. This design enabled the grafting of PNIPAAm polymer chains from the outer FhuA channel surface. In the future, the synthesized building blocks of the FhuA channel proteins and polymer chains will be used for generation of hybrid membranes for nanofiltration processes. These generated membranes can be applied in the downstream processing as molecular sieves for the separation of different components which is essential for synthesizing sweeteners, pesticides and pharmaceuticals.

Read more

 
 

In vitro flow cytometry-based screening platform for cellulase engineering

In vitro flow cytometry-based screening platform for cellulase engineering Copyright: © Bio VI

Screening technologies are of pivotal importance for tailoring biocatalysts in directed evolution, as millions of mutant enzyme variants could be generated in every trial. Hence, ultrahigh throughput screening techniques have been developed in order to still complete such trials in a reasonable amount of time. These techniques are well capable of analyzing up to 107 events per hour and thus can analyze the complete coverage of a generated protein sequence with high efficiency.

This technology becomes even more powerful if it is coupled with a cell-free enzyme expression technique. This expression method enables the experimentator to reduce diversity loss when transforming mutant libraries into expression hosts, to design enzymes of animal or human origin or even perform directed evolution of toxic enzymes.

The first ever combination of such a cell-free compartmentalization platform with a flow cytometry-based screening has been achieved in the InVitroFlow technology and successfully applied to directed evolution of cellulose enzymes.

Read more

 
 

Screening through the PLICable promoter toolbox enhances protein production in Escherichia coli

Screening through the PLICable promoter toolbox enhances protein production in Escherichia coli Copyright: © Bio VI

Escherichia coli is a common host for recombinant protein production in which product titers are highly dependent on the employed expression system. Thereby, promoters are a key element to control gene expression levels. In this study, a novel PLICable promoter toolbox was developed. It enables the identification of the most suitable promoter out of ten IPTG-inducible promoters (T7, A3, lpp, tac, pac, Sp6, lac, npr, trc and syn) for high level protein production in a single cloning step and after a screening experiment.

Read more