Materials Synthetic Biology

Long-lasting polypore fungi are significant producers of terpene cyclases of high interest for medicinal or biotechnological applications. Following the 1000 Fungal Genomes initiative launched by the Joint Genome Institute, the genome of Cubamyces (C.) menziesii and identified 18 genes encoding sesquiterpene cyclases (STCs) is explored. In a search for robust catalysts suitable for practical applications, the 18 codon-optimized open reading frames are cloned and overproduced the C. menziesii STCs in Escherichia coli. In ten cases, the catalytically active enzyme is purified and tested with three chemically synthesized linear diphosphates: geranyl diphosphate, farnesyl diphosphate (FDP), and geranylgeranyl diphosphate. Only FDP proved to be a substrate for these 10 enzymes. The product specificity of all these enzymes is determined by (GC-MS) gas chromatography mass spectrometry and (NMR) nuclear magnetic resonance analysis. Among the 10 enzymes, four produced a predominant compound, four yielded two main compounds, and the remaining two acted as a multiproduct catalysts. This work sheds light on the potential sesquiterpenes involved in the chemical ecology of the polypore C. menziesii and provides evidence for the potential of Polyporales fungi in the identification of new sesquiterpene cyclase activities.

Elastocaloric technology is a new way to heat and cool spaces by using stretchy metals, called shape-memory alloys, instead of harmful refrigerant gases. When these metals are squeezed or stretched, they heat up; and when they relax, they cool down. This process is called the elastocaloric effect and it is more energy efficient than traditional cooling systems, making it a cleaner, greener alternative. Elastocaloric systems could cool homes, schools, and workplaces, and they could refrigerate food and medicine in areas with limited electricity. Researchers are also testing this technology for cooling and heating of electric vehicles, where it could help conserve battery life, and for heating buildings in colder climates. Despite its promise, elastocaloric technology faces challenges, such as improving the durability of materials and making the shape-memory alloys more affordable. With continued research, this technology could someday help to reduce greenhouse gas emissions, lower energy costs, and bring life-saving cooling to more people all over the world.

Methods for the precise temporal control of cell surface receptor activation are indispensable for the investigation of signaling processes in mammalian cells. Optogenetics enables such precise control, but its application in primary cells is limited by the imperative for genetic manipulation of target cells. We here describe a method that overcomes this obstacle and enables the precise activation of the T cell receptor in nongenetically engineered human T cells by light. Our optogenetic receptor activation system OptoREACT employs a TCR-specific scFv fused to PIF6 that interacts with tetramerized PhyB in a light-dependent manner and thereby clusters and activates the T cell receptor in response to red light. OptoREACT not only omits genetic manipulation of the target cell but, because of its modular nature, is likely applicable to a broad range of oligomerization-activated cell surface receptors.

The construction and assembly of information-processing biomaterials are limited by the need for laborious assembly of various circuits. A new framework to assemble protein-based elements encoding complex Boolean operations enables user-defined release of biomolecules from these materials.

Molecular optogenetics allows the control of molecular signaling pathways in response to light. This enables the analysis of the kinetics of signal activation and propagation in a spatially and temporally resolved manner. A key strategy for such control is the light-inducible clustering of signaling molecules, which leads to their activation and subsequent downstream signaling. In this work, an optogenetic approach is developed for inducing graded clustering of different proteins that are fused to eGFP, a widely used protein tag. To this aim, an eGFP-specific nanobody is fused to Cryptochrome 2 variants engineered for different orders of cluster formation. This is exemplified by clustering eGFP-IKKα and eGFP-IKKβ, thereby achieving potent and reversible activation of NF-κB signaling. It is demonstrated that this approach can activate downstream signaling via the endogenous NF-κB pathway and is thereby capable of activating both an NF-κB-responsive reporter construct as well as endogenous NF-κB-responsive target genes as analyzed by RNA sequencing. The generic design of this system is likely transferable to other signaling pathways to analyze the kinetics of signal activation and propagation.

Formaldehyde is the smallest existing aldehyde, a highly reactive color less gas at room temperature and ubiquitously present in our atmosphere. Because of its reactivity leading to the crosslinking of macromolecules like proteins, it is widely used in industrial applications, but also in cell biology in order to preserve cells and tissues for further analysis. In this work, we show that formaldehyde releasing solutions commonly used for fixation of cells, can diffuse via the gas phase to the neighboring well and influence signaling processes in the therein cultured cells. To analyze this effect, we utilized a stable reporter cell line for YAP signaling or a gene expression-based reporter for activation of the NF-κB pathway. We could show that next to formaldehyde, also glutaraldehyde and acetaldehyde were able to activate those signaling pathways. Additionally, especially the stable reporter cell line based on YAP signaling can also be used as sensor for bioavailable formaldehyde, being highly sensitive, easy to use, and reversible. The observed impact of formaldehyde on cellular signaling underscores the need for careful planning of experimental protocols and emphasizes the importance of implementing proper controls when utilizing this reagent in cellular signaling analyses.

The pH dependence of the absorption and (time-resolved) fluorescence of two red-shifted fluorescent proteins, mCardinal and mNeptune, was investigated. Decay-associated spectra were measured following fluorescence excitation at 470 nm in PBS buffer with a pH that ranged from 5.5 to 8.0. The fluorescence of both proteins shows two different decay components. mCardinal exhibits an increase in the long-lived fluorescence component with acidification from 1.34 ns at pH 8.0 to 1.62 ns at pH 5.5. An additional fast decay component with 0.64 ns at pH 8.0 up to 1.1 ns at pH 5.5 was found to be blue-shifted compared to the long-lived component. The fluorescence lifetime of mNeptune is insensitive to pH. DAS of mCardinal were simulated assuming a coupled two-level system to describe the 1S state of the chromophore within two different conformations of the protein. MD simulations were conducted to correlate the experimentally observed pH-induced change in the lifetime in mCardinal with its molecular properties. While the chromophores of both protein variants are stabilized by the same number of hydrogen bonds, it was found that the chromophore in mCardinal exhibits more water contacts compared to mNeptune. In mCardinal, interaction between the chromophore and Glu-145 is reduced as compared to mNeptune, but interaction with Thr-147 which is Ser-147 in mNeptune is stronger in mCardinal. Therefore, the dynamics of the excited-state proton transfer (ESPT) might be different in mCardinal and mNeptune. The pH dependency of ESPT is suggested as a key mechanism for pH sensitivity.

Optogenetics is a versatile and powerful tool for the control and analysis of cellular signaling processes. The activation of cellular receptors by light using optogenetic switches usually requires genetic manipulation of cells. However, this considerably limits the application in primary, nonengineered cells, which is crucial for the study of physiological signaling processes and for controlling cell fate and function for therapeutic purposes. To overcome this limitation, we developed a system for the light-dependent extracellular activation of cell surface receptors of nonengineered cells termed OptoREACT (Optogenetic Receptor Activation) based on the light-dependent protein interaction of A. thaliana phytochrome B (PhyB) with PIF6. In the OptoREACT system, a PIF6-coupled antibody fragment binds the T cell receptor (TCR) of Jurkat or primary human T cells, which upon illumination is bound by clustered phytochrome B to induce receptor oligomerization and activation. For clustering of PhyB, we either used tetramerization by streptavidin or immobilized PhyB on the surface of cells to emulate the interaction of a T cell with an antigen-presenting cell. We anticipate that this extracellular optogenetic approach will be applicable for the light-controlled activation of further cell surface receptors in primary, nonengineered cells for versatile applications in fundamental and applied research.

Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

A novel approach for controlling translation initiation in mammalian cells is demonstrated based on the conditional attachment of eukaryotic translation initiation factor-binding proteins to the 3′ UTR of mRNAs via small molecule-, light-, or protein-responsive interactions. The technology overcomes limitations of previously used transcription-based switches and was shown to be functional in managing diabetes or tumor growth in preclinical animal models.