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

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

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

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences