Materials Synthetic Biology

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.

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The kinetics of a ligand-receptor interaction determine the responses of the receptor-expressing cell. One approach to experimentally and reversibly change this kinetics on demand is optogenetics. We have previously developed a system in which the interaction of a modified receptor with an engineered ligand can be controlled by light. In this system the ligand is a soluble Phytochrome B (PhyB) tetramer and the receptor is fused to a mutated PhyB-interacting factor (PIFS). However, often the natural ligand is not soluble, but expressed as a membrane protein on another cell. This allows ligand-receptor interactions in two dimensions. Here, we developed a strategy to generate cells that display PhyB as a membrane-bound protein by expressing the SpyCatcher fused to a transmembrane domain in HEK-293T cells and covalently coupling purified PhyB-SpyTag to these cells. As proof-of-principle, we use Jurkat T cells that express a GFP-PIFS-T cell receptor and show that these cells can be stimulated by the PhyB-coupled HEK-293T cells in a light dependent manner. Thus, we call the PhyB-coupled cells opto-antigen presenting cells (opto-APCs). Our work expands the toolbox of optogenetic technologies, allowing two-dimensional ligand-receptor interactions to be controlled by light.

In plants, the topological organization of membranes has mainly been attributed to the cell wall and the cytoskeleton. Additionally, few proteins, such as plant-specific remorins have been shown to function as protein and lipid organizers. Root nodule symbiosis requires continuous membrane re-arrangements, with bacteria being finally released from infection threads into membrane-confined symbiosomes. We found that mutations in the symbiosis-specific SYMREM1 gene result in highly disorganized perimicrobial membranes. AlphaFold modelling and biochemical analyses reveal that SYMREM1 oligomerizes into antiparallel dimers and may form a higher-order membrane scaffolding structure. This was experimentally confirmed when expressing this and other remorins in wall-less protoplasts is sufficient where they significantly alter and stabilize de novo membrane topologies ranging from membrane blebs to long membrane tubes with a central actin filament. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. Taken together we describe a plant-specific mechanism that allows the stabilization of large-scale membrane conformations independent of the cell wall. © 2023, The Author(s).

There are no targeted therapies for patients with triple-negative breast cancer (TNBC). TNBC is enriched in breast cancer stem cells (BCSC), which play a key role in metastasis, chemoresistance, relapse, and mortality. γδ T cells hold great potential in immunotherapy against cancer and might provide an approach to therapeutically target TNBC. γδ T cells are commonly observed to infiltrate solid tumors and have an extensive repertoire of tumor-sensing mechanisms, recognizing stress-induced molecules and phosphoantigens (pAgs) on transformed cells. Herein, we show that patient-derived triple-negative BCSCs are efficiently recognized and killed by ex vivo expanded γδ T cells from healthy donors. Orthotopically xenografted BCSCs, however, were refractory to γδ T-cell immunotherapy. We unraveled concerted differentiation and immune escape mechanisms: xenografted BCSCs lost stemness, expression of γδ T-cell ligands, adhesion molecules, and pAgs, thereby evading immune recognition by γδ T cells. Indeed, neither promigratory engineered γδ T cells, nor anti–PD-1 checkpoint blockade, significantly prolonged overall survival of tumor-bearing mice. BCSC immune escape was independent of the immune pressure exerted by the γδ T cells and could be pharmacologically reverted by zoledronate or IFNα treatment. These results pave the way for novel combinatorial immunotherapies for TNBC.

Personalized antibiotherapy ensures that the antibiotic concentration remains in the optimal therapeutic window to maximize efficacy, minimize side effects, and avoid the emergence of drug resistance due to insufficient dosing. However, such individualized schemes need frequent sampling to tailor the blood antibiotic concentrations. To optimally integrate therapeutic drug monitoring (TDM) into the clinical workflow, antibiotic levels can either be measured in blood using point-of-care testing (POCT), or can rely on noninvasive sampling. Here, a versatile biosensor with an antibody-free assay for on-site TDM is presented. The platform is evaluated with an animal study, where antibiotic concentrations are quantified in different matrices including whole blood, plasma, urine, saliva, and exhaled breath condensate (EBC). The clearance and the temporal evaluation of antibiotic levels in EBC and plasma are demonstrated. Influence of matrix effects on measured drug concentrations is determined by comparing the plasma levels with those in noninvasive samples. The system's potential for blood-based POCT is further illustrated by tracking ß‑lactam concentrations in untreated blood samples. Finally, multiplexing capabilities are explored successfully for multianalyte/sample analysis. By enabling a rapid, low-cost, sample-independent, and multiplexed on-site TDM, this system can shift the paradigm of “one‑size-fits-all” strategy. © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH

In the rapidly expanding field of molecular optogenetics, the performance of the engineered systems relies on the switching properties of the underlying genetically encoded photoreceptors. In this study, the bacterial phytochromes Cph1 and DrBphP are engineered, recombinantly produced in Escherichia coli, and characterized regarding their switching properties in order to synthesize biohybrid hydrogels with increased light-responsive stiffness modulations. The R472A mutant of the cyanobacterial phytochrome 1 (Cph1) is identified to confer the phytochrome-based hydrogels with an increased dynamic range for the storage modulus but a different light-response for the loss modulus compared to the original Cph1-based hydrogel. Stiffness measurements of human atrial fibroblasts grown on these hydrogels suggest that differences in the loss modulus at comparable changes in the storage modulus affect cell stiffness and thus underline the importance of matrix viscoelasticity on cellular mechanotransduction. The hydrogels presented here are of interest for analyzing how mammalian cells respond to dynamic viscoelastic cues. Moreover, the Cph1-R472A mutant, as well as the benchmarking of the other phytochrome variants, are expected to foster the development and performance of future optogenetic systems. © 2022 The Authors. Advanced Biology published by Wiley-VCH GmbH.

Molecular optogenetics is a highly dynamic research field. In the past two years, the field was characterized by the development of new allosteric switches as well as the forward integration of optogenetics research towards application. Further, two areas of research have significantly gathered momentum, the use of optogenetics to control liquid–liquid phase separation as well as the application of optogenetic tools in the extracellular space. Here, we review these areas and discuss future directions. © 2022 Elsevier Ltd

Clinical assessment based on a single biomarker is in many circumstances not sufficient for adequate diagnosis of a disease or for monitoring its therapy. Multiplexing, the measurement of multiple analytes from one sample and/or of the same target from different samples simultaneously, could enhance the accuracy of the diagnosis of diseases and their therapy success. Thus, there is a great and urgent demand for multiplexed biosensors allowing a low-cost, easy-to-use, and rapid on-site testing. In this work, we present a simple, flexible, and highly scalable strategy for implementing microfluidic multiplexed electrochemical biosensors (BiosensorX). Our technology is able to detect 4, 6, or 8 (different) analytes or samples simultaneously using a sequential design concept: multiple immobilization areas, where the assay components are adsorbed, followed by their individual electrochemical cells, where the amperometric signal readout takes place, within a single microfluidic channel. Here, first we compare vertical and horizontal designs of BiosensorX chips using a model assay. Owing to its easier handling and superior fluidic behavior, the vertical format is chosen as the final multiplexed chip design. Consequently, the feasibility of the BiosensorX for multiplexed on-site testing is successfully demonstrated by measuring meropenem antibiotics via an antibody-free β-lactam assay. The multiplexed biosensor platform introduced can be further extended for the simultaneous detection of other anti-infective agents and/or biomarkers (such as renal or inflammation biomarkers) as well as different (invasive and non-invasive) sample types, which would be a major step towards sepsis management and beyond. Graphical Abstract: [Figure not available: see fulltext.]. © 2022, The Author(s).