570 Biowissenschaften, Biologie
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Adaptations of Gram-Negative and Gram-Positive Probiotic Bacteria in Engineered Living Materials
(2025)
Encapsulation of microbes in natural or synthetic matrices is a key aspect of engineered living materials, although the influence of such confinement on microbial behavior is poorly understood. A few recent studies have shown that the spatial confinement and mechanical properties of the encapsulating material significantly influence microbial behavior, including growth, metabolism, and gene expression. However, comparative studies within different bacterial species under identical confinement conditions are limited. In this study, Gram-negative Escherichia coli Nissle 1917 and Gram-positive Lactiplantibacillus plantarum WCFS1 were encapsulated in hydrogel matrices, and their growth, metabolic activity, and recombinant gene expression were examined under varying degrees of hydrogel stiffness, achieved by adjusting the polymer concentration and chemical cross-linking. Both bacteria grow from single cells into confined colonies, but more interestingly, in E. coli gels, mechanical properties influenced colony growth, size, and morphology, whereas this did not occur in L. plantarum gels. However, with both bacteria, increased matrix stiffness led to higher levels of recombinant protein production within the colonies. By measuring metabolic heat from the bacterial gels using the isothermal microcalorimetry technique, it was inferred that E. coli adapts to the mechanical restrictions through multiple metabolic transitions and is significantly affected by the different hydrogel properties. Contrastingly, both of these aspects were not observed with L. plantarum. These results revealed that despite both bacteria being gut-adapted probiotics with similar geometries, mechanical confinement affects them considerably differently. The weaker influence of matrix stiffness on L. plantarum is attributed to its slower growth and thicker cell wall, possibly enabling the generation of higher turgor pressures to overcome restrictive forces under confinement. By providing fundamental insights into the interplay between mechanical forces and bacterial physiology, this work advances our understanding of how matrix properties shape bacterial behavior. The implications of these findings will aid the design of engineered living materials for therapeutic applications.
Pyrazinones are a growing family of microbial NRPS-derived natural products showing interesting biological activities. These compounds are characterized by the presence of either a di- or trisubstituted heterocyclic, nonaromatic 2(1 H)-pyrazinone core in their structure. The most commonly occurring disubstituted pyrazinone natural products are synthesized through a dipeptide intermediate, which is further cyclized to yield the pyrazinone moiety. Trisubstituted pyrazinones are seldom found in natural products, with JBIR56 and JBIR57, isolated from marine Streptomyces, being notable examples. In contrast to the simply organized disubstituted pyrazinones, JBIR56 and JBIR57 are syn-thesized as tetrapeptides with unnatural beta-amino acid residue involved in the for-mation of the pyrazinone moiety. Despite interesting structural features, biosynthetic routes leading to the production of these compounds have not been reported yet. Here we report the discovery of new members of trisubstituted pyrazinone family– tetrapeptides ichizinones A-C in Streptomyces sp. LV45-129. Through sequence analysis and heterologous expression, a biosynthetic gene cluster encoding ichizinone production was identified. Based on gene annotation and sequence homology, a biosynthetic model was suggested. The presented results provide insights into the biosynthesis of rare trisubstituted pyrazinone natural products.
The approval of Onpattro® (2018) and Comirnaty (2020) has driven interest in nanoparticulate nucleotide delivery. Newer concepts in gene therapy however, require not only the delivery of one, but multiple nucleotides. Examples are CRISPR/Cas9 gene editing and cancer immunotherapy. However, the current gold standard for nucleotide delivery – lipid nanoparticles – faces significant challenges, including limitations for co-encapsulation and nucleotide-nucleotide interactions. Aim of this study was to design a core-shell system featuring separate encapsulation of two nucleotides via a two-step formulation process. Six distinct cationic polymers were combined with three anionic polymers, resulting in 18 core compositions. Screening of these formulations identified three potent lipopolyplexes (LPPs), which were further evaluated and compared in terms of transfection efficiency, expression kinetics, storage stability, and nebulization performance. Among them, the combination of poly-L-arginine and poly-L-glutamic acid demonstrated the highest overall performance. Our systems enabled precise co-delivery of two model mRNAs in a controlled ratio, demonstrating potential for advanced therapeutic applications. Additionally, the role of mRNA localization within the LPP was investigated. Surface-loaded mRNA demonstrated superior transfection efficiency and shear resistance compared to core-loaded mRNA, which lost functionality under nebulization.
Advances in the past decades in materials science, biofabrication methods, and synthetic biology have given rise to new fields like living materials. A living material is a class of biohybrid composite with living elements, including bacteria, yeasts, fungi, and mammalian cells, integrated with nonliving components. (1−6) These materials combine the advantages of both living and nonliving components to generate novel functions such as responses to environmental parameters and syntheses of complex biomolecules. (7) The nonliving aspect combines diverse chemistries and manufacturing techniques to support or enhance the functions of the living part. (6) Living materials as therapeutics (Living Therapeutic Materials, LTMs) bring revolutionary options to diagnostic and therapeutic practice, offering a solution to life-concerning issues by life itself (Figure 1). Living Therapeutic Materials are revolutionizing classical drug delivery devices, as they can produce therapeutics long-term, in situ, and on demand. This represents a more sustainable way for treatment. To realize Living Therapeutic Materials in the clinic, more preclinical studies need to be carried out so the concerns in terms of safety are well understood and their capacity as a more efficient delivery system is assessed. In the past decade, there has been a rise in the number of proof-of-concept LTMs and yet, the preclinical investigation of these materials is just starting.
Melt Electrowriting (MEW) is a powerful technique in tissue engineering, enabling the precise fabrication of scaffolds with complex geometries. One of the most important parameters of MEW is collector speed, which has been extensively studied in relation to critical translation speed. However, its influence on crystallinity was overlooked. Crystallinity is crucial for the mechanical properties and degradation behavior of the scaffolds. Therefore, in this study, we present how printing affects the crystallinity of fibers and the resulting mechanical properties of MEW scaffolds. In systematic analysis, we observed a significant reduction in scaffold crystallinity with increased speed, as evidenced by wide-angle X-ray scattering. This decrease in crystallinity was attributed to differences in cooling rates, impacting the polycaprolactone molecular orientation within the fibers. By using tensile testing, we observed the decrease in scaffold Young's modulus with increasing collector speed. Given the relation between crystallinity and mechanical properties of the material, we developed a finite element analysis model that accounts for changes in crystallinity by employing distinct bulk Young's modulus values to help characterize scaffold mechanical behavior under tensile loading. The model reveals insights into scaffold stiffness variation with different architectural designs. These insights offer valuable guidance for optimizing 3D printing to obtain scaffolds with desired mechanical properties.
During mating in budding yeast, cells use pheromones to locate each other and fuse. This model system has shaped our current understanding of signal transduction and cell polarization in response to extracellular signals. The cell populations producing extracellular signal landscapes themselves are, however, less well understood, yet crucial for functionally testing quantitative models of cell polarization and for controlling cell behavior through bioengineering approaches. Here we engineered optogenetic control of pheromone landscapes in mating populations of budding yeast, hijacking the mating-pheromone pathway to achieve spatial control of growth, cell morphology, cell-cell fusion, and distance-dependent gene expression in response to light. Using our tool, we were able to spatially control and shape pheromone gradients, allowing the use of a biophysical model to infer the properties of large-scale gradients generated by mating populations in a single, quantitative experimental setup, predicting that the shape of such gradients depends quantitatively on population parameters. Spatial optogenetic control of diffusible signals and their degradation provides a controllable signaling environment for engineering artificial communication and cell-fate systems in gel-embedded cell populations without the need for physical manipulation.
Living bacterial therapeutics represent an exciting frontier for achieving controlled drug release within the body. However, genetic modules require improvement to control the production and release of therapeutic biomolecules in medically relevant strains. Model probiotic strains like E. coli Nissle 1917 have extensive genetic toolkits but still lack rapidly responsive and stringent genetic switches to regulate drug release. On the other hand, probiotic bacteria from the Lactobacilli family have broader applicability in the body but remain as non-model strains with restrictive genetic programmability. This thesis addresses both these limitations. Firstly, I developed a strategy to achieve strict control over the release of an enzymatically synthesized antibiotic (darobactin) from E. coli Nissle 1917. By combining parts from pre-established genetic switches, I created a thermo-amplifier circuit that released darobactin at pathogen inhibitory levels within a few hours. Secondly, I expanded the genetic toolbox of the probiotic Lactiplantibacillus plantarum WCFS1 strain with two genetic parts - a strong constitutive promoter (PtlpA) and several type II toxin-antitoxin (TA)-based plasmid retention systems. The performance of these genetic modules in recombinant plasmids was verified using reporter proteins such as mCherry and Staphylococcal nuclease without the need for antibiotic-based selection pressure.
Friction between fingertip and surface is a key contribution to tactile perception during active exploration of materials. We explore the role of skin factors such as stratum corneum thickness and hydration, deformability, elasticity, or density of sweat glands and of Meissner corpuscles in friction and tactile perception. The skin parameters were determined non-invasively for the glabrous skin at the index finger pad of 60 participants. Sets of randomly rough plastic surfaces and of micro-structured fibrillar rubber surfaces were explored as model materials with well-defined parameterized textures. Friction varies greatly between participants, and this variation can be explained to 70% by skin factors for the randomly rough plastic surfaces. The predictability of friction by skin factors is much lower for micro-structured rubber surfaces with bendable fibrils, where 50% of variance is explained for the stiffest fibrils but only 20% for the most bendable fibrils. The participants’ age is the key predictor for their tactile sensitivity to perceive the fibrils, where age is negatively correlated to the density of Meissner corpuscles. The results suggest that stratum corneum hydration, skin deformability, and age are important factors for friction and perception in active tactile exploration of materials.
Metabolite-Responsive Control of Transcription by Phase Separation-Based Synthetic Organelles
(2025)
Living natural materials have remarkable sensing abilities that translate external cues into functional changes of the material. The reconstruction of such sensing materials in bottom-up synthetic biology provides the opportunity to develop synthetic materials with life-like sensing and adaptation ability. Key to such functions are material modules that translate specific input signals into a biomolecular response. Here, we engineer a synthetic organelle based on liquid–liquid phase separation that translates a metabolic signal into the regulation of gene transcription. To this aim, we engineer the pyruvate-dependent repressor PdhR to undergo liquid–liquid phase separation in vitro by fusion to intrinsically disordered regions. We demonstrate that the resulting coacervates bind DNA harboring PdhR-responsive operator sites in a pyruvate dose-dependent and reversible manner. We observed that the activity of transcription units on the DNA was strongly attenuated following recruitment to the coacervates. However, the addition of pyruvate resulted in a reversible and dose-dependent reconstitution of transcriptional activity. The coacervate-based synthetic organelles linking metabolic cues to transcriptional signals represent a materials approach to confer stimulus responsiveness to minimal bottom-up synthetic biological systems and open opportunities in materials for sensor applications.
Photocrosslinkable formulations based on the radical thiol-ene reaction are considered better alternatives than methacrylated counterparts for light-based fabrication processes. This study quantifies differences between thiol-ene and methacrylated crosslinked hydrogels in terms of precursors stability, the control of the crosslinking process, and the resolution of printed features particularized for hyaluronic acid (HA) inks at concentrations relevant for bioprinting. First, the synthesis of HA functionalized with norbornene, allyl ether, or methacrylate groups with the same molecular weight and comparable degrees of functionalization is presented. The thiol-ene hydrogel precursors show storage stability over 15 months, 3.8 times higher than the methacrylated derivative. Photorheology experiments demonstrate up to 4.7-times faster photocrosslinking. Network formation in photoinitiated thiol-ene HA crosslinking allows higher temporal control than in methacrylated HA, which shows long post-illumination hardening. Using digital light processing, 4% w/v HA hydrogels crosslinked with a dithiol allowed printing of 13.5 × 4 × 1 mm3 layers with holes of 100 µm resolution within 2 s. This is the smallest feature size demonstrated in DLP printing with HA-based thiol-ene hydrogels. The results are important to estimate the extent to which the synthetic effort of introducing –ene functions can pay off in the printing step.
