Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin

Protein-DNA conjugates have found numerous applications in the field of diagnostics and nanobiotechnology, however, their intrinsic susceptibility to DNA degradation by nucleases represents a major obstacle for many applications. We here report the selective covalent conjugation of the protein streptavidin (STV) with phosphorothioate oligonucleotides (psDNA) containing a terminal alkylthiolgroup as the chemically addressable linking unit, using a heterobifunctional NHS-/maleimide crosslinker. The psDNA-STV conjugates were synthesized in about 10% isolated yields. We demonstrate that the terminal alkylthiol group selectively reacts with the maleimide while the backbone sulfur atoms are not engaged in chemical conjugation. The novel psDNA-STV conjugates retain their binding capabilities for both biotinylated macromolecules and the complementary nucleic acid. Moreover, the psDNA-STV conjugate retained its binding capacity for complementary oligomers even after a nuclease digestion step, which effectively degrades deoxyribonucleotide oligomers and thus the binding capability of regular DNA-STV conjugates. The psDNA-STV therefore hold particular promise for applications e.g. in proteome research and novel biosensing devices, where interfering endogenous nucleic acids need to be removed from analytes by nuclease digestion

A Modular System for the Rapid Comparison of Different Membrane Anchors for Surface Display on

Comparison of different membrane anchor motifs for the surface display of a protein of interest (passenger) is crucial for achieving the best possible performance. However, generating genetic fusions of the passenger to various membrane anchors is time-consuming. We herein employ a recently developed modular display system, in which the membrane anchor and the passenger are expressed separately and assembled in situ via SpyCatcher and SpyTag interaction, to readily combine a model passenger cytochrome P450 BM3 (BM3) with four different membrane anchors (Lpp-OmpA, PgsA, INP and AIDA-I). This approach has the significant advantage that passengers and membrane anchors can be freely combined in a modular fashion without the need to generate direct genetic fusion constructs in each case. We demonstrate that the membrane anchors impact not only cell growth and membrane integrity, but also the BM3 surface display capacity and whole-cell biocatalytic activity. The previously used Lpp-OmpA as well as PgsA were found to be efficient for the display of BM3 via SpyCatcher/SpyTag interaction. Our strategy can be transferred to other user-defined anchor and passenger combinations and could thus be used for acceleration and improvement of various applications involving cell surface display

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

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

Formulation and characterisation of enzyme-based biomaterials for μFluidic experiments

The fundamental principle of biological compartmentalisation of cellular life provides the basis for space-time resolved reaction processes. Based on this, intensive work is currently done on the use of interconnected, continuously flowing reaction chambers in order to improve the reaction control and efficiency of chemical syntheses, especially with the inclusion of biocatalysts (so called flow biocatalysis). Therefore, the Institute for Biological Interfaces IBG-1 aims at the formulation of enzyme-based biomaterials and the associated testing of novel gene-encoded coupling systems. The resulting enzyme fusions will be used as modular building blocks for the assembly of catalytically active materials, with different formulations (hydrogels or thin films) and characterized in terms of their immobilization and biocatalytic activity in miniaturized flow reactors. During the process of formulating an optimised biomaterial, we developed and established the self-assembling all-enzyme hydogels (AEHs). These protein materials consist of the two homotetrameric enzymes, (R)-selective alcohol dehydrogenase (ADH) and the cofactor regenerating glucose 1-dehydrogenase (GDH), that are genetically fused with either the SpyCatcher (SC) or the SpyTag (ST). The AEHs were characterised via dynamic light scattering (DLS) and scanning electron microscopy (SEM) in terms of physical properties. Moreover, the gels showed excellent stereoselectivity, stable conversion rates and high space-time yields (STY) for more than seven days in continuous flow experiments

Microfluidics for adaptation of microorganisms to stress: design and application

Microfluidic systems have fundamentally transformed the realm of adaptive laboratory evolution (ALE) for microorganisms by offering unparalleled control over environmental conditions, thereby optimizing mutant generation and desired trait selection. This review summarizes the substantial influence of microfluidic technologies and their design paradigms on microbial adaptation, with a primary focus on leveraging spatial stressor concentration gradients to enhance microbial growth in challenging environments. Specifically, microfluidic platforms tailored for scaled-down ALE processes not only enable highly autonomous and precise setups but also incorporate novel functionalities. These capabilities encompass fostering the growth of biofilms alongside planktonic cells, refining selection gradient profiles, and simulating adaptation dynamics akin to natural habitats. The integration of these aspects enables shaping phenotypes under pressure, presenting an unprecedented avenue for developing robust, stress-resistant strains, a feat not easily attainable using conventional ALE setups. The versatility of these microfluidic systems is not limited to fundamental research but also offers promising applications in various areas of stress resistance. As microfluidic technologies continue to evolve and merge with cutting-edge methodologies, they possess the potential not only to redefine the landscape of microbial adaptation studies but also to expedite advancements in various biotechnological areas

Imine Reductase Based All-Enzyme Hydrogel with Intrinsic Cofactor Regeneration for Flow Biocatalysis

All-enzyme hydrogels are biocatalytic materials, with which various enzymes can be immobilized in microreactors in a simple, mild, and efficient manner to be used for continuous flow processes. Here we present the construction and application of a cofactor regenerating hydrogel based on the imine reductase GF3546 from Streptomyces sp. combined with the cofactor regenerating glucose-1-dehydrogenase from Bacillus subtilis. The resulting hydrogel materials were characterized in terms of binding kinetics and viscoelastic properties. The materials were formed by rapid covalent crosslinking in less than 5 min, and they showed a typical mesh size of 67 ± 2 nm. The gels were applied for continuous flow biocatalysis. In a microfluidic reactor setup, the hydrogels showed excellent conversions of imines to amines for up to 40 h in continuous flow mode. Variation of flow rates led to a process where the gels showed a maximum space-time-yield of 150 g·(L·day)−1 at 100 μL/mi