Sustainable protein regeneration in encapsulated materials.

Zhu et al. introduce MELG (materials engineered by living grafting), combining engineered microbes with non-living scaffolds for functional protein regeneration within. These MELGs can be used for long-term controlled release, enzyme-mediated biocatalysis, and DNA purification. This approach offers enhanced functionality and durability in bioactive materials compared to traditional non-living counterparts.

Discovery of a high-performance phage-derived promoter/repressor system for probiotic lactobacillus engineering.

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.

Rheological behavior of Pluronic/Pluronic diacrylate hydrogels used for bacteria encapsulation in engineered living materials.

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.

Expanding the genetic programmability of Lactiplantibacillus plantarum.

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.

Engineered living materials for the conversion of a low-cost food-grade precursor to a high-value flavonoid.

Microbial biofactories allow the upscaled production of high-value compounds in biotechnological processes. This is particularly advantageous for compounds like flavonoids that promote better health through their antioxidant, anti-bacterial, anti-cancer and other beneficial effects but are produced in small quantities in their natural plant-based hosts. Bacteria like E. coli have been genetically modified with enzyme cascades to produce flavonoids like naringenin and pinocembrin from coumaric or cinnamic acid. Despite advancements in yield optimization, the production of these compounds still involves high costs associated with their biosynthesis, purification, storage and transport. An alternative production strategy could involve the direct delivery of the microbial biofactories to the body. In such a strategy, ensuring biocontainment of the engineered microbes in the body and controlling production rates are major challenges. In this study, these two aspects are addressed by developing engineered living materials (ELMs) consisting of probiotic microbial biofactories encapsulated in biocompatible hydrogels. Engineered probiotic E. coli Nissle 1917 able to efficiently convert cinnamic acid into pinocembrin were encapsulated in poly(vinyl alcohol)-based hydrogels. The biofactories are contained in the hydrogels for a month and remain metabolically active during this time. Control over production levels is achieved by the containment inside the material, which regulates bacteria growth, and by the amount of cinnamic acid in the medium.

Retraction: Photoactivatable Hsp47: A Tool to Regulate Collagen Secretion and Assembly.

Adv. Sci. 2019, 6, 1801982 DOI: 10.1002/advs.201801982 The above article, published online on May 3, 2019, in Wiley Online Library (https://doi.org/10.1002/advs.201801982), has been retracted by agreement between the authors, the journal Editor-in-Chief Kirsten Severing, and Wiley-VCH GmbH. The retraction has been agreed on following concerns raised by a third party and a subsequent investigation by the corresponding authors. Data depicted in Figure 4 and Figure 5 could not be reproduced in follow-up experiments. Therefore, the conclusions associated with those figures in the article are considered invalid. E.S.K. participated in the study design, performed measurements, analyzed the data, compiled the figures and participated in manuscript writing. A.d.C. and S.S. participated in the study design, research supervision, and manuscript writing. J.I.P. participated in the study design. M.K.L.H. participated in research supervision and manuscript revision. C.M. assisted with the experimental procedures and data collection.

In vitro evaluation of immune responses to bacterial hydrogels for the development of living therapeutic materials.

In living therapeutic materials (LTMs), organisms genetically programmed to produce and deliver drugs are encapsulated in porous matrices acting as physical barriers between the therapeutic organisms and the host cells. LTMs consisting of engineered E. coli encapsulated in Pluronic F127-based hydrogels have been frequently used in LTM designs but their immunogenicity has not been tested. In this study, we investigate the response of human peripheral blood mononuclear cells (PBMCs) exposed to this bacteria/hydrogel combination. The release of inflammation-related cytokines and cytotoxic proteins and the subsets of natural killer cells and T cells were examined. Encapsulation of the bacteria in hydrogels considerably lowers their immunogenicity. ClearColi, an endotoxin-free variant of E. coli, did not polarize NK cells into the more cytolytic CD16dim subset as E. coli. Our results demonstrate that ClearColi-encapsulated hydrogels generate low immunogenic response and are suitable candidates for the development of LTMs for in vivo testing to assess a potential clinical use. Nevertheless, we observed a stronger immune response (elevated levels of IFNγ, IL-6 and cytotoxic proteins) in pro-inflammatory PBMCs characterized by a high spontaneous release of IL-2. This highlights the need to identify recipients who have a higher likelihood of experiencing undesired immune responses to LTMs with IL-2 serving as a potential predictive marker. Additionally, including anti-inflammatory measures in living therapeutic material designs could be beneficial for such recipients.

Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli.

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.

Plasmonic stimulation of gold nanorods for the photothermal control of engineered living materials.

Engineered living materials (ELMs) encapsulate microorganisms within polymeric matrices for biosensing, drug delivery, capturing viruses, and bioremediation. It is often desirable to control their function remotely and in real time and so the microorganisms are often genetically engineered to respond to external stimuli. Here, we combine thermogenetically engineered microorganisms with inorganic nanostructures to sensitize an ELM to near infrared light. For this, we use plasmonic gold nanorods (AuNR) that have a strong absorption maximum at 808 nm, a wavelength where human tissue is relatively transparent. These are combined with Pluronic-based hydrogel to generate a nanocomposite gel that can convert incident near infrared light into heat locally. We perform transient temperature measurements and find a photothermal conversion efficiency of 47 %. Steady-state temperature profiles from local photothermal heating are quantified using infrared photothermal imaging and correlated with measurements inside the gel to reconstruct spatial temperature profiles. Bilayer geometries are used to combine AuNR and bacteria-containing gel layers to mimic core-shell ELMs. The thermoplasmonic heating of an AuNR-containing hydrogel layer that is exposed to infrared light diffuses to the separate but connected hydrogel layer with bacteria and stimulates them to produce a fluorescent protein. By tuning the intensity of the incident light, it is possible to activate either the entire bacterial population or only a localized region.

In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1.

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.

Encapsulation of bacteria in bilayer Pluronic thin film hydrogels: A safe format for engineered living materials.

In engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.

Regulating Bacterial Behavior within Hydrogels of Tunable Viscoelasticity.

Engineered living materials (ELMs) are a new class of materials in which living organism incorporated into diffusive matrices uptake a fundamental role in material's composition and function. Understanding how the spatial confinement in 3D can regulate the behavior of the embedded cells is crucial to design and predict ELM's function, minimize their environmental impact and facilitate their translation into applied materials. This study investigates the growth and metabolic activity of bacteria within an associative hydrogel network (Pluronic-based) with mechanical properties that can be tuned by introducing a variable degree of acrylate crosslinks. Individual bacteria distributed in the hydrogel matrix at low density form functional colonies whose size is controlled by the extent of permanent crosslinks. With increasing stiffness and elastic response to deformation of the matrix, a decrease in colony volumes and an increase in their sphericity are observed. Protein production follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that matrix design can be used to control and regulate the composition and function of ELMs containing microorganisms. Interestingly, design parameters for matrices to regulate bacteria behavior show similarities to those elucidated for 3D culture of mammalian cells.

Exogenous supply of Hsp47 triggers fibrillar collagen deposition in skin cell cultures in vitro.

BACKGROUND: Collagen is a structural protein that provides mechanical stability and defined architectures to skin. In collagen-based skin disorders this stability is lost, either due to mutations in collagens or in the chaperones involved in collagen assembly. This leads to chronic wounds, skin fragility, and blistering. Existing approaches to treat such conditions rely on administration of small molecules to simulate collagen production, like 4-phenylbutyrate (4-PBA) or growth factors like TGF-ß. However, these molecules are not specific for collagen synthesis, and result in unsolicited side effects. Hsp47 is a collagen-specific chaperone with a major role in collagen biosynthesis. Expression levels of Hsp47 correlate with collagen deposition. This article explores the stimulation of collagen deposition by exogenously supplied Hsp47 (collagen specific chaperone) to skin cells, including specific collagen subtypes quantification. RESULTS: Here we quantify the collagen deposition level and the types of deposited collagens after Hsp47 stimulation in different in vitro cultures of cells from human skin tissue (fibroblasts NHDF, keratinocytes HaCat and endothelial cells HDMEC) and mouse fibroblasts (L929 and MEF). We find upregulated deposition of fibrillar collagen subtypes I, III and V after Hsp47 delivery. Network collagen IV deposition was enhanced in HaCat and HDMECs, while fibril-associated collagen XII was not affected by the increased intracellular Hsp47 levels. The deposition levels of fibrillar collagen were cell-dependent i.e. Hsp47-stimulated fibroblasts deposited significantly higher amount of fibrillar collagen than Hsp47-stimulated HaCat and HDMECs. CONCLUSIONS: A 3-fold enhancement of collagen deposition was observed in fibroblasts upon repeated dosage of Hsp47 within the first 6 days of culture. Our results provide fundamental understanding towards the idea of using Hsp47 as therapeutic protein to treat collagen disorders.

Photoactivatable Hsp47: A Tool to Regulate Collagen Secretion and Assembly.

Collagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen-specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability, and intracellular transport of procollagen triple helices. This work reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. Photoactivatable Hsp47 contains a non-natural light-responsive tyrosine (o-nitro benzyl tyrosine (ONBY)) at Tyr383 position of the protein sequence. This mutation renders Hsp47 inactive toward collagen binding. The inactive, photoactivatable protein is easily uptaken by cells within a few minutes of incubation, and accumulated at the endoplasmic reticulum via retrograde KDEL receptor-mediated uptake. Upon light exposure, the photoactivatable Hsp47 turns into functional Hsp47 in situ. The increased intracellular concentration of Hsp47 results in stimulated secretion of collagen. The ability to promote collagen synthesis on demand, with spatiotemporal resolution, and in diseased state cells is demonstrated in vitro. It is envisioned that photoactivatable Hsp47 allows unprecedented fundamental studies of collagen biosynthesis, matrix biology, and inspires new therapeutic concepts in biomedicine and tissue regeneration.

Optoregulated Protein Release from an Engineered Living Material.

Developing materials to encapsulate and deliver functional proteins inside the body is a challenging yet rewarding task for therapeutic purposes. High production costs, mostly associated with the purification process, short-term stability in vivo, and controlled and prolonged release are major hurdles for the clinical application of protein-based biopharmaceuticals. In an attempt to overcome these hurdles, herein, the possibility of incorporating bacteria as protein factories into a material and externally controlling protein release using optogenetics is demonstrated. By engineering bacteria to express and secrete a red fluorescent protein in response to low doses of blue light irradiation and embedding them in agarose hydrogels, living materials are fabricated capable of releasing proteins into the surrounding medium when exposed to light. These bacterial hydrogels allow spatially confined protein expression and dosed protein release over several weeks, regulated by the area and extent of light exposure. The possibility of incorporating such complex functions in a material using relatively simple material and genetic engineering strategies highlights the immense potential and versatility offered by living materials for protein-based biopharmaceutical delivery.

Optoregulated Drug Release from an Engineered Living Material: Self-Replenishing Drug Depots for Long-Term, Light-Regulated Delivery.

On-demand and long-term delivery of drugs are common requirements in many therapeutic applications, not easy to be solved with available smart polymers for drug encapsulation. This work presents a fundamentally different concept to address such scenarios using a self-replenishing and optogenetically controlled living material. It consists of a hydrogel containing an active endotoxin-free Escherichia coli strain. The bacteria are metabolically and optogenetically engineered to secrete the antimicrobial and antitumoral drug deoxyviolacein in a light-regulated manner. The permeable hydrogel matrix sustains a viable and functional bacterial population and permits diffusion and delivery of the synthesized drug to the surrounding medium at quantities regulated by light dose. Using a focused light beam, the site for synthesis and delivery of the drug can be freely defined. The living material is shown to maintain considerable levels of drug production and release for at least 42 days. These results prove the potential and flexibility that living materials containing engineered bacteria can offer for advanced therapeutic applications.

Toward Light-Regulated Living Biomaterials.

Living materials are an emergent material class, infused with the productive, adaptive, and regenerative properties of living organisms. Property regulation in living materials requires encoding responsive units in the living components to allow external manipulation of their function. Here, an optoregulated Escherichia coli (E. coli)-based living biomaterial that can be externally addressed using light to interact with mammalian cells is demonstrated. This is achieved by using a photoactivatable inducer of gene expression and bacterial surface display technology to present an integrin-specific miniprotein on the outer membrane of an endotoxin-free E. coli strain. Hydrogel surfaces functionalized with the bacteria can expose cell adhesive molecules upon in situ light-activation, and trigger cell adhesion. Surface immobilized bacteria are able to deliver a fluorescent protein to the mammalian cells with which they are interacting, indicating the potential of such a bacterial material to deliver molecules to cells in a targeted manner.

Cell Adhesion on RGD-Displaying Knottins with Varying Numbers of Tryptophan Amino Acids to Tune the Affinity for Assembly on Cucurbit[8]uril Surfaces.

Cell adhesion is studied on multivalent knottins, displaying RGD ligands with a high affinity for integrin receptors, that are assembled on CB[8]-methylviologen-modified surfaces. The multivalency in the knottins stems from the number of tryptophan amino acid moieties, between 0 and 4, that can form a heteroternary complex with cucurbit[8]uril (CB[8]) and surface-tethered methylviologen (MV2+). The binding affinity of the knottins with CB[8] and MV2+ surfaces was evaluated using surface plasmon resonance spectroscopy. Specific binding occurred, and the affinity increased with the valency of tryptophans on the knottin. Additionally, increased multilayer formation was observed, attributed to homoternary complex formation between tryptophan residues of different knottins and CB[8]. Thus, we were able to control the surface coverage of the knottins by valency and concentration. Cell experiments with mouse myoblast (C2C12) cells on the self-assembled knottin surfaces showed specific integrin recognition by the RGD-displaying knottins. Moreover, cells were observed to elongate more on the supramolecular knottin surfaces with a higher valency, and in addition, more pronounced focal adhesion formation was observed on the higher-valency knottin surfaces. We attribute this effect to the enhanced coverage and the enhanced affinity of the knottins in their interaction with the CB[8] surface. Collectively, these results are promising for the development of biomaterials including knottins via CB[8] ternary complexes for tunable interactions with cells.

Cell Adhesion on Dynamic Supramolecular Surfaces Probed by Fluid Force Microscopy-Based Single-Cell Force Spectroscopy.

Biomimetic and stimuli-responsive cell-material interfaces are actively being developed to study and control various cell-dynamics phenomena. Since cells naturally reside in the highly dynamic and complex environment of the extracellular matrix, attempts are being made to replicate these conditions in synthetic biomaterials. Supramolecular chemistry, dealing with noncovalent interactions, has recently provided possibilities to incorporate such dynamicity and responsiveness in various types of architectures. Using a cucurbit[8]uril-based host-guest system, we have successfully established a dynamic and electrochemically responsive interface for the display of the integrin-specific ligand, Arg-Gly-Asp (RGD), to promote cell adhesion. Due to the weak nature of the noncovalent forces by which the components at the interface are held together, we expected that cell adhesion would also be weaker in comparison to traditional interfaces where ligands are usually immobilized by covalent linkages. To assess the stability and limitations of our noncovalent interfaces, we performed single-cell force spectroscopy studies using fluid force microscopy. This technique enabled us to measure rupture forces of multiple cells that were allowed to adhere for several hours on individual substrates. We found that the rupture forces of cells adhered to both the noncovalent and covalent interfaces were nearly identical for up to several hours. We have analyzed and elucidated the reasons behind this result as a combination of factors including the weak rupture force between linear Arg-Gly-Asp and integrin, high surface density of the ligand, and increase in effective concentration of the supramolecular components under spread cells. These characteristics enable the construction of highly dynamic biointerfaces without compromising cell-adhesive properties.