Sandra Kröll

Karlsruhe Institute of Technology

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Christof M. Niemeyer

Karlsruhe Institute of Technology

Kersten S. Rabe

Karlsruhe Institute of Technology

Patrick Bitterwolf

Karlsruhe Institute of Technology

Julian Hertel

Karlsruhe Institute of Technology

Christof Wöll

Karlsruhe Institute of Technology

Tilo Baumbach

Karlsruhe Institute of Technology

Maximilian Grösche

Karlsruhe Institute of Technology

M. S. Zubér

Karlsruhe Institute of Technology

Annika J. Weber

Karlsruhe Institute of Technology

Parvesh Wadhwani

Karlsruhe Institute of Technology

Cooperative Institutions

Karlsruhe Institute of Technology

Interface (United States)

FIZ Karlsruhe – Leibniz Institute for Information Infrastructure

Karlsruhe University of Education

Deutsches Elektronen-Synchrotron DESY

Stanford Synchrotron Radiation Lightsource

FZI Research Center for Information Technology

University of Duisburg-Essen

University of Tübingen

Case Western Reserve University

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Biocatalytic Foams from Microdroplet‐Formulated Self‐Assembling Enzymes

Advanced Materials (2023)

Julian Hertel Patrick Bitterwolf Sandra Kröll Astrid Winterhalter Annika J. Weber

Industrial biocatalysis plays an important role in the development of a sustainable economy, as enzymes can be used to synthesize an enormous range of complex molecules under environmentally friendly conditions. To further develop the field, intensive research is being conducted on process technologies for continuous flow biocatalysis in order to immobilize large quantities of enzyme biocatalysts in microstructured flow reactors under conditions that are as gentle as possible in order to realize efficient material conversions. Here, monodisperse foams consisting almost entirely of enzymes covalently linked via SpyCatcher/SpyTag conjugation are reported. The biocatalytic foams are readily available from recombinant enzymes via microfluidic air-in-water droplet formation, can be directly integrated into microreactors, and can be used for biocatalytic conversions after drying. Reactors prepared by this method show surprisingly high stability and biocatalytic activity. The physicochemical characterization of the new materials is described and exemplary applications in biocatalysis are shown using two-enzyme cascades for the stereoselective synthesis of chiral alcohols and the rare sugar tagatose.

Biocatalysis

Microreactor

Environmentally Friendly

Dispersity

10.1002/adma.202303952

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Citations (11)

Nucleic Acid‐based Enzyme Cascades—Current Trends and Future Perspectives

Angewandte Chemie International Edition (2023)

Sandra Kröll Christof M. Niemeyer

Abstract The natural micro‐ and nanoscale organization of biomacromolecules is a remarkable principle within living cells, allowing for the control of cellular functions by compartmentalization, dimensional diffusion and substrate channeling. In order to explore these biological mechanisms and harness their potential for applications such as sensing and catalysis, molecular scaffolding has emerged as a promising approach. In the case of synthetic enzyme cascades, developments in DNA nanotechnology have produced particularly powerful scaffolds whose addressability can be programmed with nanometer precision. In this minireview, we summarize recent developments in the field of biomimetic multicatalytic cascade reactions organized on DNA nanostructures. We emphasize the impact of the underlying design principles like DNA origami, efficient strategies for enzyme immobilization, as well as the importance of experimental design parameters and theoretical modeling. We show how DNA nanostructures have enabled a better understanding of diffusion and compartmentalization effects at the nanometer length scale, and discuss the challenges and future potential for commercial applications.

10.1002/anie.202314452

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Nucleic Acid‐based Enzyme Cascades—Current Trends and Future Perspectives

Angewandte Chemie (2023)

Sandra Kröll Christof M. Niemeyer

Compartmentalization (fire protection)

DNA nanotechnology

Synthetic Biology

DNA origami

10.1002/ange.202314452

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Nano‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

Small (2023)

Sandra Kröll Teresa Burgahn Kersten S. Rabe Matthias Franzreb Christof M. Niemeyer

Abstract Artificial reconstruction of naturally evolved principles, such as compartmentalization and cascading of multienzyme complexes, offers enormous potential for the development of biocatalytic materials and processes. Due to their unique addressability at the nanoscale, DNA origami nanostructures (DON) have proven to be an exceptionally powerful tool for studying the fundamental processes in biocatalytic cascades. To systematically investigate the diffusion‐reaction network of (co)substrate transfer in enzyme cascades, a model system of stereoselective ketoreductase (KRED) with cofactor regenerating enzyme is assembled in different spatial arrangements on DNA nanostructures and is located in the sphere of microbeads (MB) as a spatially confining nano‐ and microenvironment, respectively. The results, obtained through the use of highly sensitive analytical methods, Western blot‐based quantification of the enzymes, and mass spectrometric (MS) product detection, along with theoretical modeling, provide strong evidence for the presence of two interacting compartments, the diffusion layers around the microbead and the DNA scaffold, which influence the catalytic efficiency of the cascade. It is shown that the microscale compartment exerts a strong influence on the productivity of the cascade, whereas the nanoscale arrangement of enzymes has no influence but can be modulated by the insertion of a diffusion barrier.

Microscale chemistry

Cascade reaction

10.1002/smll.202304578

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Orthogonal protein decoration of DNA nanostructures based on SpyCatcher–SpyTag interaction

Chemical Communications (2022)

Sandra Kröll Leonie Schneider Parvesh Wadhwani Kersten S. Rabe Christof M. Niemeyer

We present an efficient and readily applicable strategy for the covalent ligation of proteins to DNA origami by using the SpyCatcher-SpyTag (SC-ST) connector system. This approach showed orthogonality with other covalent connectors and has been used exemplarily for the immobilization and study of stereoselective ketoreductases to gain insight into the spatial arrangement of enzymes on DNA nanostructures.

Orthogonality

DNA origami

10.1039/d2cc05335g

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An Orthogonal Covalent Connector System for the Efficient Assembly of Enzyme Cascades on DNA Nanostructures

Small (2021)

Sandra Kröll Kersten S. Rabe Christof M. Niemeyer

Combining structural DNA nanotechnology with the virtually unlimited variety of enzymes offers unique opportunities for generating novel biocatalytic devices. However, the immobilization of enzymes is still restricted by a lack of efficient covalent coupling techniques. The rational re-engineering of the genetically fusible SNAP-tag linker is reported here. By replacing five amino acids that alter the electrostatic properties of the SNAP_R5 variant, up to 11-fold increased coupling efficiency with benzylguanine-modified oligonucleotides and DNA origami nanostructures (DON) was achieved, resulting in typical occupancy densities of 75%. The novel SNAP_R5 linker can be combined with the equally efficient Halo-based oligonucleotide binding tag (HOB). Since both linkers exhibit neither cross-reactivity nor non-specific binding, they allowed orthogonal assembly of an enzyme cascade consisting of the stereoselective ketoreductase Gre2p and the cofactor-regenerating isocitrate dehydrogenase on DON. The cascade showed approximately 1.6-fold higher activity in a stereoselective cascade reaction than the corresponding free solubilized enzymes. The connector system presented here and the methods used to validate it represent important tools for further development of DON-based multienzyme systems to investigate mechanistic effects of substrate channeling and compartmentalization relevant for exploitation in biosensing and catalysis.

Linker

DNA origami

Protein Engineering

10.1002/smll.202105095

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Intracellular Assembly of Interacting Enzymes Yields Highly‐Active Nanoparticles for Flow Biocatalysis

Chemistry - A European Journal (2022)

Patrick Bitterwolf Ahmed E. Zoheir Julian Hertel Sandra Kröll Kersten S. Rabe

All-enzyme hydrogel (AEH) particles with a hydrodynamic diameter of up to 120 nm were produced intracellularly with an Escherichia coli-based in vivo system. The inCell-AEH nanoparticles were generated from polycistronic vectors enabling simultaneous expression of two interacting enzymes, the Lactobacillus brevis alcohol dehydrogenase (ADH) and the Bacillus subtilis glucose-1-dehydrogenase (GDH), fused with a SpyCatcher or SpyTag, respectively. Formation of inCell-AEH was analyzed by dynamic light scattering and atomic force microscopy. Using the stereoselective two-step reduction of a prochiral diketone substrate, we show that the inCell-AEH approach can be advantageously used in whole-cell flow biocatalysis, by which flow reactors could be operated for >4 days under constant substrate perfusion. More importantly, the inCell-AEH concept enables the recovery of efficient catalyst materials for stable flow bioreactors in a simple and economical one-step procedure from crude bacterial lysates. We believe that our method will contribute to further optimization of sustainable biocatalytic processes.

Biocatalysis

10.1002/chem.202202157

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Citations (6)