A Critical View on the Use of DNA Hydrogels in Cell-Free Protein Synthesis.

Numerous studies have reported in the past that the use of protein-encoding DNA hydrogels as templates for cell-free protein synthesis (CFPS) leads to better yields than the use of conventional templates such as plasmids or PCR fragments. Systematic investigation of different types of bulk materials from pure DNA hydrogels and DNA hydrogel composites using a commercially available CFPS kit showed no evidence of improved expression efficiency. However, protein-coding DNA hydrogels were advantageously used in microfluidic reactors as immobilized templates for repetitive protein production, suggesting that DNA-based materials offer potential for future developments in high-throughput profiling or rapid in situ characterization of proteins.

Modeling-Assisted Design of Thermostable Benzaldehyde Lyases from Rhodococcus erythropolis for Continuous Production of α-Hydroxy Ketones.

Enantiopure α-hydroxy ketones are important building blocks of active pharmaceutical ingredients (APIs), which can be produced by thiamine-diphosphate-dependent lyases, such as benzaldehyde lyase. Here we report the discovery of a novel thermostable benzaldehyde lyase from Rhodococcus erythropolis R138 (ReBAL). While the overall sequence identity to the only experimentally confirmed benzaldehyde lyase from Pseudomonas fluorescens Biovar I (PfBAL) was only 65 %, comparison of a structural model of ReBAL with the crystal structure of PfBAL revealed only four divergent amino acids in the substrate binding cavity.

3D-Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4-Hydroxystilbene.

Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio- and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose-based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor-free phenacrylate decarboxylase enzymes suitable for the production of 4-vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk-shaped agarose-based modules that can be used for compartmentalized flow microreactors.

Direct coupling analysis improves the identification of beneficial amino acid mutations for the functional thermostabilization of a delicate decarboxylase.

The optimization of enzyme properties for specific reaction conditions enables their tailored use in biotechnology. Predictions using established computer-based methods, however, remain challenging, especially regarding physical parameters such as thermostability without concurrent loss of activity. Employing established computational methods such as energy calculations using FoldX can lead to the identification of beneficial single amino acid substitutions for the thermostabilization of enzymes.