Bioprogrammable Materials

Engineered living materials (ELMs) made of bacteria in hydrogels have shown considerable promise for therapeutic applications through controlled and sustained release of complex biopharmaceuticals at low costs and with reduced wastage. While most therapeutic ELMs use E. coli due to its large genetic toolbox, most live biotherapeutic bacteria in development are lactic acid bacteria due to native health benefits they offer. Among these, lactobacilli form the largest family of probiotics with therapeutic potential in almost all sites of the body with a microbiome. A major factor limiting the use of lactobacilli in ELMs is their limited genetic toolbox. This study expands on recent work to expand the genetic programmability of probiotic Lactiplantibacillus plantarum WCFS1 for protein secretion and encapsulate it in a simple, cost-effective, and biocompatible core–shell alginate bead to develop an ELM. The controlled release of recombinant proteins is demonstrated, even up to 14 days from this ELM, thereby terming it PEARL – Protein Eluting Alginate with Recombinant Lactobacilli. Notably, lactobacillus encapsulation offered benefits like bacterial containment, protein release profile stabilization, and metabolite-induced cytotoxicity prevention. These findings demonstrate the mutual benefits of combining recombinant lactobacilli with alginate for the controlled and sustained release of proteins.

Engineered living materials (ELMs) rely on the ability to control cell behavior in material systems. ELMs containing bacteria secreting beneficial molecules are being developed for therapeutic purposes. Using commensal strains embedded in physically cross-linked agarose hydrogels, we systematically investigate how gel rigidity and initial bacterial density affect the morphology of bacterial colonies and their secretory function. Although often considered independently, these parameters jointly define the microscale environment experienced by embedded cells, influencing nutrient access, mechanical interactions, and potential cell-to-cell communication. We show that matrix rigidity effectively tunes aggregate morphology, modulating their shape and compactness, without compromising bacterial growth or secretion. In parallel, initial bacterial density determines the biomass accumulation dynamics and spatial distribution of aggregates, which in turn influence the onset and temporal profile of secretory activity, without altering its final magnitude. This decoupling between structural organization and secretory output opens new possibilities for engineering ELMs with tailored architectures and prolonged secretory and release activity.

Engineered living materials (ELMs), which usually comprise bacteria, fungi, or animal cells entrapped in polymeric matrices, offer limitless possibilities in fields like drug delivery or biosensing. Determining the conditions that sustain ELM performance while ensuring compatibility with ELM hosts is essential before testing them in vivo. This is critical to reduce animal experimentation and can be achieved through in vitro investigations. Currently, there are no standards that ensure ELM compatibility with host tissues. Towards this goal, we designed a 96-well plate-based screening method to streamline ELM growth across culture conditions and determine their compatibility potential in vitro. We showed proliferation of three bacterial species encapsulated in hydrogels over time and screened six different cell culture media. We fabricated ELMs in bilayer and monolayer formats and tracked bacterial leakage as a measure of ELM biocontainment. After screening, an appropriate medium was selected that sustained growth of an ELM, and it was used to study cytocompatibility in vitro. ELM cytotoxicity on murine fibroblasts and human monocytes was studied by adding ELM supernatants and measuring cell membrane integrity and live/dead staining, respectively, proving ELM cytocompatibility. Our work illustrates a simple setup to streamline the screening of compatible environmental conditions of ELMs with the host.

Living therapeutic and diagnostic materials based on engineered microorganisms are emerging as a novel approach with the perspective of providing patient-tailored, sustainable, and cost-effective healthcare solutions. In this review, we focus on recent advances in using genetically or chemically engineered microorganisms as living diagnostics, therapeutics, and as a means of prevention for various diseases. We also highlight the applications of living therapeutics for acute and chronic diseases, and the role of micro/macro-encapsulation of the engineered microorganisms. We further showcase the current success of engineered living therapeutics in clinical trials and discuss challenges and future trends in the field.

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.

Living therapeutics are attractive candidates to tackle the limitations of classically delivered therapeutic peptides, which are often poorly stable and require cost-intensive modifications. Their functional assessment is limited to animal experiments, which increase the complexity to evaluate the dynamic nature of these systems. Therefore, we developed an in vitro model of endotoxemia using macrophages to assess early-stage anti-inflammatory Living therapeutics. We refined the model based on three anti-inflammatory peptides (KCF-18, I6P7, and α-MSH) and identified suitable therapeutic concentrations and treatment durations. We applied the model to Lactiplantibacillus plantarum TF103, a probiotic engineered to secrete these peptides. The model revealed that Living therapeutics enhanced the effects of the peptides, requiring lower amounts of anti-inflammatory effects. This points to potential synergistic effects between peptides and bacteria. The model presented here allows the investigation of dynamic regimes, which could be useful in the development of complex systems such as the ones encountered in Living therapeutics.

Living microbial therapeutics promise precise, programmable interventions at disease sites, yet most demonstrations of on demand drug release still rely on Escherichia coli, whose rich genetic toolkit is unmatched among probiotics. In particular, genetic parts to regulate in situ protein production are severely lacking in non-model probiotic bacteria like lactobacilli. Here, we equip the probiotic Lactiplantibacillus plantarum with high-performance genetic switches and show how material encapsulation can further enhance their behavior. By integrating cumate or vanillate-responsive operators and repressors with the strongest constitutive promoter in L. plantarum (Ptec), we generated two switches that support micromolar range induction. In rapidly growing culture conditions, acidification-associated leakiness of the switch was observed, which could compromise their applicability for precise on-demand delivery of drugs. Furthermore, such leakiness also limits the duration for which these engineered probiotics can be reliably used. By restricting growth through mild temperature or nutrient limitation, acidification and leakiness were suppressed. Strikingly, immobilizing the engineered cells in core-shell alginate beads (Protein Eluting Alginate with Recombinant Lactobacilli, PEARLs) almost eliminated leakiness, enabling day-scale, reversible control of intracellular reporters and secreted enzymes. This leakiness suppression persisted when two strains carrying orthogonal switches were co-encapsulated and even after miniaturization to submillimeter beads. These results expand the genetic toolbox of probiotic L. plantarum, demonstrate the synergy between genetic circuit design and material encapsulation, and advance lactobacilli toward stimuli-responsive therapeutic platforms.

Pluronic (Plu) hydrogels mixed with variable fractions of Pluronic diacrylate (PluDA) have become popular matrices to encapsulate bacteria and control their growth in engineered living materials. Here we study the rheological response of 30 wt.% Plu/PluDA hydrogels with PluDA fraction between 0 and 1. We quantify the range of viscoelastic properties that can be covered in this system by varying in the PluDA fraction. We present stress relaxation and creep-recovery experiments and describe the variation of the critical yield strain/stress, relaxation and recovery parameters of Plu/PluDA hydrogels as function of the covalent crosslinking degree using the Burgers and Weilbull models. The analyzed hydrogels present two stress relaxations with different timescales which can be tuned with the covalent crosslinking degree. We expect this study to help users of Plu/PluDA hydrogels to estimate the mechanical properties of their systems, and to correlate them with the behaviour of bacteria in future Plu/PluDA devices of similar composition.

Background: The Lactobacillaceae family comprises many species of great importance for the food and healthcare industries, with numerous strains identified as beneficial for humans and used as probiotics. Hence, there is a growing interest in engineering these probiotic bacteria as live biotherapeutics for animals and humans. However, the genetic parts needed to regulate gene expression in these bacteria remain limited compared to model bacteria like E. coli or B. subtilis. To address this deficit, in this study, we selected and tested several bacteriophage-derived genetic parts with the potential to regulate transcription in lactobacilli.
Results: We screened genetic parts from 6 different lactobacilli-infecting phages and identified one promoter/repressor system with unprecedented functionality in Lactiplantibacillus plantarum WCFS1. The phage-derived promoter was found to achieve expression levels nearly 9-fold higher than the previously reported strongest promoter in this strain and the repressor was able to almost completely repress this expression by reducing it nearly 500-fold.
Conclusions: The new parts and insights gained from their engineering will enhance the genetic programmability of lactobacilli for healthcare and industrial applications.

Lactobacilli are ubiquitous in nature and symbiotically provide health benefits for countless organisms including humans, animals and plants. They are vital for the fermented food industry and are being extensively explored for healthcare applications. For all these reasons, there is considerable interest in enhancing and controlling their capabilities through the engineering of genetic modules and circuits. One of the most robust and reliable microbial chassis for these synthetic biology applications is the widely used Lactiplantibacillus plantarum species. However, the genetic toolkit needed to advance its applicability remains poorly equipped. This mini-review highlights the genetic parts that have been discovered to achieve food-grade recombinant protein production and speculates on lessons learned from these studies for L. plantarum engineering. Furthermore, strategies to identify, create and optimize genetic parts for real-time regulation of gene expression and enhancement of biosafety are also suggested.