Biological compartmentalization is a fundamental principle of life that allows cells to metabolize, propagate, or communicate with their environment. Much research is devoted to understanding this basic principle and to harness biomimetic compartments and catalytic cascades as tools for technological processes. This Review summarizes the current state-of-the-art of these developments, with a special emphasis on length scales, mass transport phenomena, and molecular scaffolding approaches, ranging from small cross-linkers over proteins and nucleic acids to colloids and patterned surfaces. We conclude that the future exploration and exploitation of these complex systems will largely benefit from technical solutions for the integrated, machine-assisted development and maintenance of a next generation of biotechnological processes. These goals should be achievable by implementing microfluidics, robotics, and added manufacturing techniques supplemented by theoretical simulations as well as computer-aided process modeling based on big data obtained from multiscale experimental analyses.

Structural DNA nanotechnology [1,2] and the technique of DNA origami [3] enable the rapid generation of a plethora of complex self-assembled nanostructures. [4][5][6] Since DNA molecules themselves display limited chemical, optical, and electronic functionality, it is of utmost importance to devise methods to decorate DNA scaffolds with functional moieties to realize applications in sensing, catalysis, and device fabrication. Protein functionalization is particulary desirable because it allows exploitation of an almost unlimited variety of functional elements which nature has evolved over billions of years.[7] The delicate architecture of proteins has resulted in no generally applicable method being currently available to selectively couple these components on DNA scaffolds, and thus approaches used so far are based on reversible antibodyantigen interactions, [8,9] aptamer binding, [10,11] nucleic acid hybridization of DNA-tagged proteins, [12,13] or predominantly biotin-streptavidin (STV) interactions. [14][15][16][17][18][19] We demonstrate here that DNA nanostructures can be site-specifically decorated with several different proteins by using coupling systems orthogonal to the biotin-STV system. In particular, benzylguanine (BG) and chlorohexane (CH) groups incorporated in DNA origami have been used as suicide ligands for the site-specific coupling of fusion proteins containing the self-labeling protein tags O 6 -alkylguanine-DNA-alkyltransferase (hAGT), which is often referred to as "Snap-tag", [20] or haloalkane dehalogenase, which is also known as "HaloTag".[21] By using various model proteins we demonstrate the general applicability of this approach for the generation of DNA superstructures that are selectively decorated with multiple different proteins.To realize orthogonal protein immobilization on DNA origami using self-ligating protein tags, we chose the Snap-tag, developed by Johnsson and co-workers, [20] and the commercially available HaloTag [21] system. The respective smallmolecule suicide tags (O 6 -benzylguanine (BG) and 5-chlorohexane (CH)) for both self-labeling protein tags are readily available as amino-reactive N-hydroxysuccinimide (NHS) derivatives (BG-NHS and CH-NHS; Figure 1 a). Complete derivatization of alkylamino-modified oligonucleotides was achieved by coupling with 30 molar equivalents of BG-NHS or CH-NHS, as indicated by electrophoretic analysis (Figure 1 b). To gain access to fusion proteins bearing the complementary Snap-and Halo-protein tags, we constructed expression plasmids by genetic fusion of the genes encoding the protein of interest (POI) and Snap-tag or HaloTag (see the Supporting Information). As model POIs we chose the fluorescent proteins enhanced yellow fluorescent protein (EYFP) [22] and mKate, [23] the enzymes cytochrome C peroxidase (CCP) [24] and esterase 2 from Alicyclobacillus acidocaldarius thermos (EST2), [25] to which the self-labeling tags were fused at the C terminus (POI-Snap or POI-Halo, respectively). In addition, the bispecific Halo-Snap fusion protein...

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The establishment of microfluidic enzyme cascades is a topical field of research and development, which is currently hampered by the lack of methodologies for mild and efficient immobilization of isolated enzymes. We here describe the use of self-immobilizing fusion enzymes for the modular configuration of microfluidic packed-bed reactors. Specifically, three different enzymes, the (R)-selective alcohol dehydrogenase LbADH, the (S)-selective methylglyoxal reductase Gre2p and the NADP­(H) regeneration enzyme glucose 1-dehydrogenase GDH, were genetically fused with streptavidin binding peptide, Spy and Halo-based tags, to enable their specific and directional immobilization on magnetic microbeads coated with complementary receptors. The enzyme-modified beads were loaded in four-channel microfluidic chips to create compartments that have the capability for either (R)- or (S)-selective reduction of the prochiral CS-symmetrical substrate 5-nitrononane-2,8-dione (NDK). Analysis of the isomeric hydroxyketone and diol products by chiral HPLC was used to quantitatively characterize the performance of reactors configured with different amounts of the enzymes. Long operating times of up to 14 days indicated stable enzyme immobilization and the general robustness of the reactor. Even more important, by fine-tuning of compartment size and loading, the overall product distribution could be controlled to selectively produce a single meso diol with nearly quantitative conversion (>95%) and excellent stereoselectivity (d.r. > 99:1) in a continuous flow process. We believe that our concept will be expandable to a variety of other biocatalytic or chemo-enzymatic cascade reactions.

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