Top-Down and Bottom-Up Assembly of compartmentalized Enzyme Cascades – Towards Experiments and Simulation on multiple Scales

Nature has evolved efficient principles to control a complex network of chemical reactions within living cells. These unique principles include biological compartmentalization and cascading, where for example catalytically active components are organized into spatially defined multienzyme complexes. The understanding of these networks and their transfer to in vitro systems provides an extraordinary potential of innovation for the next generation of modular production processes in industrial biocatalysis.1 On the micro- to centimeter length scale, enzymes can be incorporated into microfluidic devices to harness the precise controllability of reaction parameters for the continuous production of value-added molecules. To this end, we have developed methodologies for the mild and efficient immobilization of recombinant enzymes that are genetically encoded with fusion tags, such as Halo-tag or Snap-tag. Using this approach, stereoselective ketoreductases were integrated as self-immobilizing biocatalysts in microfluidic packed-bed reactors.2 In an effort to gain deeper insights into the enzymatic mechanisms and reactor behavior, we are combing experimental data acquisition with mathematical modeling.3 With this approach, we demonstrate an increased biocatalytic process efficiency along with the reduction of material and costs. ... mehrFurthermore, to fully exploit the concept of enzyme cascades and to understand diffusion and mass transport processes on the nanometer length scale, it is essential to have accurate control over stoichiometry and spacing of multiple interacting enzymes. We demonstrate that supramolecular fabrication of such well-defined enzyme assemblies can be achieved by using concepts of structural DNA nanotechnology.4