Periplasmic stress contributes to a trade-off between protein secretion and cell growth in Escherichia coli Nissle 1917.

Maximizing protein secretion is an important target in the design of engineered living systems. In this paper, we characterize a trade-off between cell growth and per-cell protein secretion in the curli biofilm secretion system of Escherichia coli Nissle 1917. Initial characterization using 24-h continuous growth and protein production monitoring confirms decreased growth rates at high induction, leading to a local maximum in total protein production at intermediate induction. Propidium iodide (PI) staining at the endpoint indicates that cellular death is a dominant cause of growth reduction. Assaying variants with combinatorial constructs of inner and outer membrane secretion tags, we find that diminished growth at high production is specific to secretory variants associated with periplasmic stress mediated by outer membrane secretion and periplasmic accumulation of protein containing the outer membrane transport tag. RNA sequencing experiments indicate upregulation of known periplasmic stress response genes in the highly secreting variant, further implicating periplasmic stress in the growth-secretion trade-off. Overall, these results motivate additional strategies for optimizing total protein production and longevity of secretory engineered living systems Graphical Abstract.

Single domain antibodies against enteric pathogen virulence factors are active as curli fiber fusions on probiotic E. coli Nissle 1917.

Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic E. coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eukaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.

A single-cell nanocoating of probiotics for enhanced amelioration of antibiotic-associated diarrhea.

The gut microbiota represents a large community of microorganisms that play an important role in immune regulation and maintenance of homeostasis. Living bacteria receive increasing interest as potential therapeutics for gut disorders, because they inhibit the colonization of pathogens and positively regulate the composition of bacteria in gut. However, these treatments are often accompanied by antibiotic administration targeting pathogens. In these cases, the efficacy of therapeutic bacteria is compromised by their susceptibility to antibiotics. Here, we demonstrate that a single-cell coating composed of tannic acids and ferric ions, referred to as 'nanoarmor', can protect bacteria from the action of antibiotics. The nanoarmor protects both Gram-positive and Gram-negative bacteria against six clinically relevant antibiotics. The multiple interactions between the nanoarmor and antibiotic molecules allow the antibiotics to be effectively absorbed onto the nanoarmor. Armored probiotics have shown the ability to colonize inside the gastrointestinal tracts of levofloxacin-treated rats, which significantly reduced antibiotic-associated diarrhea (AAD) resulting from the levofloxacin-treatment and improved some of the pre-inflammatory symptoms caused by AAD. This nanoarmor strategy represents a robust platform to enhance the potency of therapeutic bacteria in the gastrointestinal tracts of patients receiving antibiotics and to avoid the negative effects of antibiotics in the gastrointestinal tract.

The living interface between synthetic biology and biomaterial design.

Recent far-reaching advances in synthetic biology have yielded exciting tools for the creation of new materials. Conversely, advances in the fundamental understanding of soft-condensed matter, polymers and biomaterials offer new avenues to extend the reach of synthetic biology. The broad and exciting range of possible applications have substantial implications to address grand challenges in health, biotechnology and sustainability. Despite the potentially transformative impact that lies at the interface of synthetic biology and biomaterials, the two fields have, so far, progressed mostly separately. This Perspective provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between the two communities. We highlight the near-term applications of this interface to the development of hierarchically structured biomaterials, from bioinspired building blocks to 'living' materials that sense and respond based on the reciprocal interactions between materials and embedded cells.

Simple Affinity-Based Method for Concentrating Viruses from Wastewater Using Engineered Curli Fibers.

Wastewater surveillance is a proven method for tracking community spread and prevalence of some infectious viral diseases. A primary concentration step is often used to enrich viral particles from wastewater prior to subsequent viral quantification and/or sequencing. Here, we present a simple procedure for concentrating viruses from wastewater using bacterial biofilm protein nanofibers known as curli fibers. Through simple genetic engineering, we produced curli fibers functionalized with single-domain antibodies (also known as nanobodies) specific for the coat protein of the model virus bacteriophage MS2. Using these modified fibers in a simple spin-down protocol, we demonstrated efficient concentration of MS2 in both phosphate-buffered saline (PBS) and in the wastewater matrix. Additionally, we produced nanobody-functionalized curli fibers capable of binding the spike protein of SARS-CoV-2, showing the versatility of the system. Our concentration protocol is simple to implement, can be performed quickly under ambient conditions, and requires only components produced through bacterial culture. We believe this technology represents an attractive alternative to existing concentration methods and warrants further research and optimization for field-relevant applications.

Programmable microbial ink for 3D printing of living materials produced from genetically engineered protein nanofibers.

Living cells have the capability to synthesize molecular components and precisely assemble them from the nanoscale to build macroscopic living functional architectures under ambient conditions. The emerging field of living materials has leveraged microbial engineering to produce materials for various applications but building 3D structures in arbitrary patterns and shapes has been a major challenge. Here we set out to develop a bioink, termed as "microbial ink" that is produced entirely from genetically engineered microbial cells, programmed to perform a bottom-up, hierarchical self-assembly of protein monomers into nanofibers, and further into nanofiber networks that comprise extrudable hydrogels. We further demonstrate the 3D printing of functional living materials by embedding programmed Escherichia coli (E. coli) cells and nanofibers into microbial ink, which can sequester toxic moieties, release biologics, and regulate its own cell growth through the chemical induction of rationally designed genetic circuits. In this work, we present the advanced capabilities of nanobiotechnology and living materials technology to 3D-print functional living architectures.

Hybrid Living Capsules Autonomously Produced by Engineered Bacteria.

Bacterial cellulose (BC) has excellent material properties and can be produced sustainably through simple bacterial culture, but BC-producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli (E. coli). Here, a simple approach is reported for producing highly programmable BC materials through incorporation of engineered E. coli. The acetic acid bacterium Gluconacetobacter hansenii is cocultured with engineered E. coli in droplets of glucose-rich media to produce robust cellulose capsules, which are then colonized by the E. coli upon transfer to selective lysogeny broth media. It is shown that the encapsulated E. coli can produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, capsules are produced which can alter their own bulk physical properties through enzyme-induced biomineralization. This novel system uses a simple fabrication process, based on the autonomous activity of two bacteria, to significantly expand the functionality of BC-based living materials.

Robust Self-Regeneratable Stiff Living Materials Fabricated from Microbial Cells.

Living systems have not only the exemplary capability to fabricate materials (e.g. wood, bone) under ambient conditions but they also consist of living cells that imbue them with properties like growth and self-regeneration. Like a seed that can grow into a sturdy living wood, we wondered: can living cells alone serve as the primary building block to fabricate stiff materials? Here we report the fabrication of stiff living materials (SLMs) produced entirely from microbial cells, without the incorporation of any structural biopolymers (e.g. cellulose, chitin, collagen) or biominerals (e.g. hydroxyapatite, calcium carbonate) that are known to impart stiffness to biological materials. Remarkably, SLMs are also lightweight, strong, resistant to organic solvents and can self-regenerate. This living materials technology can serve as a powerful biomanufacturing platform to design and develop advanced structural and cellular materials in a sustainable manner.

Water-processable, biodegradable and coatable aquaplastic from engineered biofilms.

Petrochemical-based plastics have not only contaminated all parts of the globe, but are also causing potentially irreversible damage to our ecosystem because of their non-biodegradability. As bioplastics are limited in number, there is an urgent need to design and develop more biodegradable alternatives to mitigate the plastic menace. In this regard, we report aquaplastic, a new class of microbial biofilm-based biodegradable bioplastic that is water-processable, robust, templatable and coatable. Here, Escherichia coli was genetically engineered to produce protein-based hydrogels, which are cast and dried under ambient conditions to produce aquaplastic, which can withstand strong acid/base and organic solvents. In addition, aquaplastic can be healed and welded to form three-dimensional architectures using water. The combination of straightforward microbial fabrication, water processability and biodegradability makes aquaplastic a unique material worthy of further exploration for packaging and coating applications.

Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium, commonly employed to treat certain gastrointestinal disorders. It is fast emerging as an important target for the development of therapeutic engineered bacteria, benefiting from the wealth of knowledge of E. coli biology and ease of manipulation. Bacterial synthetic biology projects commonly utilize engineered plasmid vectors, which are simple to engineer and can reliably achieve high levels of protein expression. However, plasmids typically require antibiotics for maintenance, and the administration of an antibiotic is often incompatible with in vivo experimentation or treatment. EcN natively contains plasmids pMUT1 and pMUT2, which have no known function but are stable within the bacteria. Here, we describe the development of the pMUT plasmids into a robust platform for engineering EcN for in vivo experimentation, alongside a CRISPR-Cas9 system to remove the native plasmids. We systematically engineered both pMUT plasmids to contain selection markers, fluorescent markers, temperature sensitive expression, and curli secretion systems to export a customizable functional material into the extracellular space. We then demonstrate that the engineered plasmids were maintained in bacteria as the engineered bacteria pass through the mouse GI tract without selection, and that the secretion system remains functional, exporting functionalized curli proteins into the gut. Our plasmid system presents a platform for the rapid development of therapeutic EcN bacteria.

Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut.

Mucosal healing plays a critical role in combatting the effects of inflammatory bowel disease, fistulae and ulcers. While most treatments for such diseases focus on systemically delivered anti-inflammatory drugs, often leading to detrimental side effects, mucosal healing agents that target the gut epithelium are underexplored. We genetically engineer Escherichia coli Nissle 1917 (EcN) to create fibrous matrices that promote gut epithelial integrity in situ. These matrices consist of curli nanofibers displaying trefoil factors (TFFs), known to promote intestinal barrier function and epithelial restitution. We confirm that engineered EcN can secrete the curli-fused TFFs in vitro and in vivo, and is non-pathogenic. We observe enhanced protective effects of engineered EcN against dextran sodium sulfate-induced colitis in mice, associated with mucosal healing and immunomodulation. This work lays a foundation for the development of a platform in which the in situ production of therapeutic protein matrices from beneficial bacteria can be exploited.

Towards the directed evolution of protein materials.

Protein-based materials have emerged as a powerful instrument for a new generation of biological materials, with many chemical and mechanical capabilities. Through the manipulation of DNA, researchers can design proteins at the molecular level, engineering a vast array of structural building blocks. However, our capability to rationally design and predict the properties of such materials is limited by the vastness of possible sequence space. Directed evolution has emerged as a powerful tool to improve biological systems through mutation and selection, presenting another avenue to produce novel protein materials. In this prospective review, we discuss the application of directed evolution for protein materials, reviewing current examples and developments that could facilitate the evolution of protein for material applications.

Genetically Programmable Self-Regenerating Bacterial Hydrogels.

A notable challenge for the design of engineered living materials (ELMs) is programming a cellular system to assimilate resources from its surroundings and convert them into macroscopic materials with specific functions. Here, an ELM that uses Escherichia coli as its cellular chassis and engineered curli nanofibers as its extracellular matrix component is demonstrated. Cell-laden hydrogels are created by concentrating curli-producing cultures. The rheological properties of the living hydrogels are modulated by genetically encoded factors and processing steps. The hydrogels have the ability to grow and self-renew when placed under conditions that facilitate cell growth. Genetic programming enables the gels to be customized to interact with different tissues of the gastrointestinal tract selectively. This work lays a foundation for the application of ELMs with therapeutic functions and extended residence times in the gut.

Congo Red Fluorescence for Rapid In Situ Characterization of Synthetic Curli Systems.

Curli are amyloid proteins that are assembled into extracellular polymeric fibers by bacteria during biofilm formation. The beta-sheet-rich protein CsgA, the primary structural component of the fibers, is secreted through dedicated machinery and self-assembles into cell-anchored fibers many times longer than the cell. Here, we have developed an in situ fluorescence assay for curli production that exploits the fluorescent properties of Congo red (CR) dye when bound to amyloid, allowing for rapid and robust curli quantification. We initially evaluated three amyloid-binding dyes for the fluorescent detection of curli in bacterial culture and found only Congo red compatible with in situ quantification. We further characterized the fluorescent properties of the dye directly in bacterial culture and calibrated the fluorescence using purified CsgA protein. We then used the Congo red assay to rapidly develop and characterize inducible curli-producing constructs in both an MC4100-derived lab strain of Escherichia coli and a derivative of the probiotic strain E. coli Nissle. This technique can be used to evaluate curli production in a minimally invasive manner using a range of equipment, simplifying curli quantification and the development of novel engineered curli systems.IMPORTANCE Curli are proteins produced by many bacteria as a structural component of biofilms, and they have recently emerged as a platform for fabrication of biological materials. Curli fibers are very robust and resistant to degradation, and the curli subunits can tolerate many protein fusions, facilitating the biosynthesis of novel functional materials. A serious bottleneck in the development of more sophisticated engineered curli systems is the rapid quantification of curli production by the bacteria. In this work we address this issue by developing a technique to monitor curli production directly in bacterial cultures, allowing for rapid curli quantification in a manner compatible with many powerful high-throughput techniques that can be used to engineer complex biological material systems.

Light-driven fine chemical production in yeast biohybrids.

Inorganic-biological hybrid systems have potential to be sustainable, efficient, and versatile chemical synthesis platforms by integrating the light-harvesting properties of semiconductors with the synthetic potential of biological cells. We have developed a modular bioinorganic hybrid platform that consists of highly efficient light-harvesting indium phosphide nanoparticles and genetically engineered Saccharomyces cerevisiae, a workhorse microorganism in biomanufacturing. The yeast harvests photogenerated electrons from the illuminated nanoparticles and uses them for the cytosolic regeneration of redox cofactors. This process enables the decoupling of biosynthesis and cofactor regeneration, facilitating a carbon- and energy-efficient production of the metabolite shikimic acid, a common precursor for several drugs and fine chemicals. Our work provides a platform for the rational design of biohybrids for efficient biomanufacturing processes with higher complexity and functionality.

Biomimetic engineering of conductive curli protein films.

Bioelectronic systems derived from peptides and proteins are of particular interest for fabricating novel flexible, biocompatible and bioactive devices. These synthetic or recombinant systems designed for mediating electron transport often mimic the proteinaceous appendages of naturally occurring electroactive bacteria. Drawing inspiration from such conductive proteins with a high content of aromatic residues, we have engineered a fibrous protein scaffold, curli fibers produced by Escherichia coli bacteria, to enable long-range electron transport. We report the genetic engineering and characterization of curli fibers containing aromatic residues of different nature, with defined spatial positioning, and with varying content on single self-assembling CsgA curli subunits. Our results demonstrate the impressive versatility of the CsgA protein for genetically engineering protein-based materials with new functions. Through a scalable purification process, we show that macroscopic gels and films can be produced, with engineered thin films exhibiting a greater conductivity compared with wild-type curli films. We anticipate that this engineered conductive scaffold, and our approach that combines computational modeling, protein engineering, and biosynthetic manufacture will contribute to the improvement of a range of useful bio-hybrid technologies.

Tracking of Engineered Bacteria In Vivo Using Nonstandard Amino Acid Incorporation.

The rapidly growing field of microbiome research presents a need for better methods of monitoring gut microbes in vivo with high spatial and temporal resolution. We report a method of tracking microbes in vivo within the gastrointestinal tract by programming them to incorporate nonstandard amino acids (NSAA) and labeling them via click chemistry. Using established machinery constituting an orthogonal translation system (OTS), we engineered Escherichia coli to incorporate p-azido-l-phenylalanine (pAzF) in place of the UAG (amber) stop codon. We also introduced a mutant gene encoding for a cell surface protein (CsgA) that was altered to contain an in-frame UAG codon. After pAzF incorporation and extracellular display, the engineered strains could be covalently labeled via copper-free click reaction with a Cy5 dye conjugated to the dibenzocyclooctyl (DBCO) group. We confirmed the functionality of the labeling strategy in vivo using a murine model. Labeling of the engineered strain could be observed using oral administration of the dye to mice several days after colonization of the gastrointestinal tract. This work sets the foundation for the development of in vivo tracking microbial strategies that may be compatible with noninvasive imaging modalities and are capable of longitudinal spatiotemporal monitoring of specific microbial populations.

Thermal Transition of Bimetallic Metal-Phenolic Networks to Biomass-Derived Hierarchically Porous Nanofibers.

The development and utilization of biomass resources could contribute to new materials for long-term sustainable energy storage and environmental applications, reduce environmental impacts, and meet the urgent need for green and sustainable development strategies. Herein, a bimetallic metal-phenolic network (MPN) was applied to incorporate different metallic element species into cattle skin and fabricate collagen-fiber-derived complex oxide nanofibers using natural polyphenols (Myrica tannins). Direct thermal transition of these biomass-MPN composites generates hierarchically porous nanofibers possessing micro- and mesoporous architectures along with a well-preserved macroscopic structure. The pore system and complex oxide composition provide excellent photocatalytic performance. This low-cost, simple, and readily scalable MPN-based approach provides a straightforward route to synthesize nanostructured materials directly from biomass, which could play important roles in a wide range of potential applications.

Modulating bacterial and gut mucosal interactions with engineered biofilm matrix proteins.

Extracellular appendages play a significant role in mediating communication between bacteria and their host. Curli fibers are a class of bacterial fimbria that is highly amenable to engineering. We demonstrate the use of engineered curli fibers to rationally program interactions between bacteria and components of the mucosal epithelium. Commensal E. coli strains were engineered to produce recombinant curli fibers fused to the trefoil family of human cytokines. Biofilms formed from these strains bound more mucins than those producing wild-type curli fibers, and modulated mucin rheology as well. When treated with bacteria producing the curli-trefoil fusions mammalian cells behaved identically in terms of their migration behavior as when they were treated with the corresponding soluble trefoil factors. Overall, this demonstrates the potential utility of curli fibers as a scaffold for the display of bioactive domains and an untapped approach to rationally modulating host-microbe interactions using bacterial matrix proteins.