Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, as antidepressants, and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from simple precursors (acetaldehyde and nitroalkenes), using a tailor-made highly enantioselective artificial “Michaelase” (4-oxalocrotonate tautomerase mutant L8Y/M45Y/F50A), an aldehyde dehydrogenase with a broad non-natural substrate scope, and a cofactor recycling system. We also report a three-step chemoenzymatic cascade route for the efficient chemical reduction of enzymatically prepared γ-nitrobutyric acids into GABA analogues in one pot, achieving high enantiopurity (e.r. up to 99:1) and high overall yields (up to 70%). This chemoenzymatic methodology offers a step-economic alternative route to important pharmaceutically active GABA analogues, and highlights the exciting opportunities available for combining chemocatalysts, natural enzymes, and designed artificial biocatalysts in multistep syntheses.

The small engineered luciferase NanoLuc has rapidly become a powerful tool in the fields of biochemistry, chemical biology, and cell biology due to its exceptional brightness and stability. The continuously expanding NanoLuc toolbox has been employed in applications ranging from biosensors to molecular and cellular imaging, and currently includes split complementation variants, engineering techniques for spectral tuning, and bioluminescence resonance energy transfer-based concepts. In this review, we provide an overview of state-of-the-art NanoLuc-based sensors and switches with a focus on the underlying protein engineering approaches. We discuss the advantages and disadvantages of various strategies with respect to sensor sensitivity, modularity, and dynamic range of the sensor and provide a perspective on future strategies and applications.

The increasing number of enzyme applications in chemical synthesis calls for new engineering methods to develop the biocatalysts of the future. An interesting concept in enzyme engineering is the generation of large‐scale mutational data in order to chart protein mutability landscapes. These landscapes allow the important discrimination between beneficial mutations and those that are neutral or detrimental, thus providing detailed insight into sequence–function relationships. As such, mutability landscapes are a powerful tool with which to identify functional hotspots at any place in the amino acid sequence of an enzyme. These hotspots can be used as targets for combinatorial mutagenesis to yield superior enzymes with improved catalytic properties, stability, or even new enzymatic activities. The generation of mutability landscapes for multiple properties of one enzyme provides the exciting opportunity to select mutations that are beneficial either for one or for several of these properties. This review presents an overview of the recent advances in the construction of mutability landscapes and discusses their importance for enzyme engineering.

Aromatic β-hydroxyaldehydes, 1,3-diols, and α,β-unsaturated aldehydes are valuable precursors to biologically active natural products and drug molecules. Herein we report the biocatalytic aldol condensation of acetaldehyde with various aromatic aldehydes to give a number of aromatic α,β-unsaturated aldehydes using a previously engineered variant of 4oxalocrotonate tautomerase [4-OT(M45T/F50A)] as carboligase. Moreover, an efficient one-pot two-step chemoenzymatic route toward chiral aromatic 1,3-diols has been developed. This one-pot chemoenzymatic strategy successfully combined a highly enantioselective aldol addition step catalyzed by a proline-based carboligase [4-OT(M45T/F50A) or TAUT015] with a chemical reduction step to convert enzymatically prepared aromatic βhydroxyaldehydes into the corresponding 1,3-diols with high optical purity (e.r. up to >99:1) and in good isolated yield (51−92%). These developed (chemo)enzymatic methodologies offer alternative synthetic choices to prepare a variety of important drug precursors.