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.

Self-Assembling All-Enzyme Hydrogels for Flow Biocatalysis.

Continuous flow biocatalysis is an emerging field of industrial biotechnology that uses enzymes immobilized in flow channels for the production of value-added chemicals. We describe the construction of self-assembling all-enzyme hydrogels that are comprised of two tetrameric enzymes. The stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerize under physiological conditions into porous hydrogels. Mounted in microfluidic reactors, the gels show excellent stereoselectivity with near quantitative conversion in the reduction of prochiral ketones along with high robustness under process and storage conditions. The gels function as compartment that retains intermediates thus enabling high total turnover numbers of the expensive cofactor NADP(H).

Biocatalytic Foams from Microdroplet-Formulated Self-Assembling Enzymes.

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.

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/min.

A Phenolic Acid Decarboxylase-Based All-Enzyme Hydrogel for Flow Reactor Technology.

Carrier-free enzyme immobilization techniques are an important development in the field of efficient and streamlined continuous synthetic processes using microreactors. Here, the use of monolithic, self-assembling all-enzyme hydrogels is expanded to phenolic acid decarboxylases. This provides access to the continuous flow production of p-hydroxystyrene from p-coumaric acid for more than 10 h with conversions ≥98% and space time yields of 57.7 g·(d·L)-1. Furthermore, modulation of the degree of crosslinking in the hydrogels resulted in a defined variation of the rheological behavior in terms of elasticity and mesh size of the corresponding materials. This work is addressing the demand of sustainable strategies for defunctionalization of renewable feedstocks.

Valency engineering of monomeric enzymes for self-assembling biocatalytic hydrogels.

All-enzyme hydrogels are efficient reagents for continuous flow biocatalysis. These materials can be obtained by self-assembly of two oligomeric enzymes, modified with the complementary SpyTag and SpyCatcher units. To facilitate access to the large proportion of biocatalytically relevant monomeric enzymes, we demonstrate that the tagging valency of the monomeric (S)-stereoselective ketoreductase Gre2p from Saccharomyces cerevisiae can be designed to assemble stable, active hydrogels with the cofactor-regenerating glucose 1-dehydrogenase GDH from Bacillus subtilis. Mounted in microfluidic reactors, these gels revealed high conversion rates and stereoselectivity in the reduction of prochiral methylketones under continuous flow for more than 8 days. The sequential use as well as parallelization by 'numbering up' of the flow reactor modules demonstrate that this approach is suitable for syntheses on the semipreparative scale.