M.Sc. Marc Blanch Asensio

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.

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.

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.

Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to their complex and thick cell walls limiting our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 µg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.

Lactobacilli are ubiquitous in nature, often beneficially associated with animals as commensals and probiotics, and are extensively used in food fermentation. Due to this close-knit association, there is considerable interest to engineer them for healthcare applications in both humans and animals, for which high-performance and versatile genetic parts are greatly desired. For the first time, we describe two genetic modules in Lactiplantibacillus plantarum that achieve high-level gene expression using plasmids that can be retained without antibiotics, bacteriocins or genomic manipulations. These include (i) a promoter, PtlpA, from a phylogenetically distant bacterium, Salmonella typhimurium, which drives up to 5-fold higher level of gene expression compared to previously reported promoters and (ii) multiple toxin-antitoxin systems as a self-contained and easy-to-implement plasmid retention strategy that facilitates the engineering of tuneable transient genetically modified organisms. These modules and the fundamental factors underlying their functionality that are described in this work will greatly contribute to expanding the genetic programmability of lactobacilli for healthcare applications.