Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

The interaction of sclerostin (Scl) with the low-density lipoprotein receptor-related protein 4 (LRP4) leads to a marked reduction in bone formation by inhibiting the Wnt/ß-catenin pathway. To characterize the Scl-LRP4 binding interface, we sorted a combinatorial library of Scl variants and isolated variants with reduced affinity to LRP4. We identified Scl single-mutation variants enriched during the sorting process and verified their reduction in affinity toward LRP4-a reduction that was not a result of changes in the variants' secondary structure or stability. We found that Scl positions K75 (loop 1) and V136 (loop 3) are critical hotspots for binding to LRP4. Our findings establish the foundation for targeting these hotspots for developing novel therapeutic strategies to promote bone formation.

Basic Guide for Approaching Drug Delivery with Extracellular Vesicles.

Extracellular vesicles (EVs) are natural carriers of biomolecules that play a crucial role in cell-to-cell communication and tissue homeostasis under normal and pathological conditions, including inflammatory diseases and cancer. Since the discovery of the pro-regenerative and immune-modulating properties of EVs, EV-based therapeutics have entered clinical trials for conditions such as myocardial infarction and autoimmune diseases, among others. Due to their unique advantages-such as superior bioavailability, substantial packaging capacity, and the ability to traverse biological barriers-EVs are regarded as a promising platform for targeted drug delivery. However, achieving a sufficient accumulation of therapeutic agents at the target site necessitates a larger quantity of EVs per dose compared to using EVs as standalone drugs. This challenge can be addressed by administering larger doses of EVs, increasing the drug dosage per administration, or enhancing the selective accumulation of EVs at target cells. In this review, we will discuss methods to improve the isolation and purification of EVs, approaches to enhance cargo packaging-including proteins, RNAs, and small-molecule drugs-and technologies for displaying targeting ligands on the surface of EVs to facilitate improved targeting. Ultimately, this guide can be applied to the development of novel classes of EV-based therapeutics and to overcoming existing technological challenges.

Chitinase and Deacetylase-Based Chitin-Degrading Bacteria: One-Pot Cascade Bioconversion of Chitin to Chitooligosaccharides.

Cascade conversion of chitin into soluble and functional chitooligosaccharides has gained great attention. However, the biotransformation route is still limited to the low catalytic performances of chitin deacetylases (CDAs) and complicated procedures. In this study, a CDA from Arthrobacter sp. Jub115 (ArCDA) was identified and characterized, which showed a higher catalytic stability than the reported CDAs, with residual activity of 80.49%, 71.12%, and 56.09% after incubation at 30, 35, and 40 °C for 24 h, respectively. Additionally, ArCDA was identified to have a broad substrate spectrum toward ß-chitin and N-acetyl chitooligosaccharides. Moreover, an engineered chitin-degrading bacteria (CDB) with cell-surface-displayed deacetylase ArCDA and chitinase SaChiB was constructed to simplify catalysis procedures, facilitating the chitobiose production of 294.30 ± 16.43 mg/L in 10 h. This study not only identified a CDA with the desirable catalytic performance but also provided a strategy for constructing CDB, facilitating the high-value utilization of chitin.

Eliminated high lipid inhibition in the anaerobic digestion of food waste for biomethane production by engineered E. coli with cell surface display lipase.

Food waste (FW) with high content of lipid typically inhibits anaerobic digestion (AD) and methane production. In this study, a novel whole-cell catalyst was created to degrade lipid by displaying lipase on the E. coli cells surface to improve FW anaerobic digestion. The methane production rose, going from 25.78 to 161.77 mL/g VS, with a greater VS removal rate of 66.3% compared to CK group (29.6%). Long-chain fatty acids (LCFAs) was similarly reduced from 1733.6 mg/L to 337 mg/L. Microbial community analysis showed the relative abundance of Acinetbacter and Hydrogenophaga were increased from 1.7% to 6.6% and 1.3%-4.9%, respectively for substrates degradation. The methanogenic Methanosarcina increased from 24.7% to 52.3% for methane production. This study provided a potential approach that might be used to lessen lipid inhibition and improve anaerobic digestion of food waste.

[Surface display and application of cadmium-binding protein CADR on the cell wall of Chlamydomonas reinhardtii].

Algae has been proven to have the potential to be efficient biosorbents in the detection and remediation of heavy metal pollution such as cadmium in the environment. This study aims to enhance the cadmium adsorption capacity of Chlamydomonas reinhardtii by expressing the cadmium-binding protein CADR on the cell wall by the surface display technology. Firstly, the golden gate technique was employed to construct the transformation vector PET-X-CADR, which anchored CADR to the cell wall with the cell wall protein GP1. The high-throughput screening with the fluorescence signal of the fusion tag YFP resulted in three engineered algal strains with high expression of CADR on the cell wall. Physiological experiments demonstrated that the CADR displayed on the cell wall did not affect the growth of the engineered algal strains exposed to cadmium with the concentration below 200 µmol/L. In the presence of 200 µmol/L cadmium, the growth rates of CADR-engineered algal strains were three times as that of the wild-type, indicating stronger tolerance of the CADR-engineered algae to cadmium. The protein lyase GLE released during the mating of Chlamydomonas was used to isolate the cell walls of wild-type and engineered strains, the cadmium content of which was compared. The results showed that the cell wall of the engineered strain exhibited an increase of 33% in cadmium adsorption capacity. This study gives insights into the application of algae in the management of cadmium pollution in the environment, especially in the recycling of heavy metals from the environment.

Engineering a high-sugar tolerant strain of Saccharomyces cerevisiae for efficient trehalose production using a cell surface display approach.

Trehalose production via a one-step enzymatic route using trehalose synthase (TreS) holds significant promise for industrial-scale applications due to its simplicity and utilization of low-cost substrates. However, the development of a robust whole-cell biocatalyst expressing TreS remains crucial for enabling practical and economically viable production. In this study, a high-sugar tolerant strain of S. cerevisiae was screened and employed as a host cell for the cell surface display of TreS from Acidiplasma aeolicum. The resultant strain, S. cerevisiae I3A, exhibited remarkable surface displayed TreS activity of 3358 U/g CDW and achieved approximately 64% trehalose yield (10.8 g/L/h productivity) from maltose. Interestingly, no glucose by-product was observed during trehalose production. The S. cerevisiae I3A cells exhibited reusability for up to 12 cycles leading to potential cost reduction of trehalose products. Therefore, our study demonstrated the development of a high-sugar tolerant S. cerevisiae strain expressing TreS on its surface as a whole-cell biocatalyst for efficient and economical trehalose production with potential applications in the food and pharmaceutical industries.

Laccase surface-display for environmental tetracycline removal: From structure to function.

Facing the increasingly prominent tetracycline pollution and the resulting environmental problems, how to find environmental and efficient treatment means is one of the current research hotspots. In this study, the laccase surface-display technology for tetracycline treatment was investigated. Via study, the type of anchoring protein had a minor influence on the laccase ability, while the type of laccase showed a major impact. Bacillus subtilis spore coat protein (CotA) exhibited higher laccase activity, stability, and efficiency in degrading tetracycline than Pleurotus ostreatus laccase 6 (Lacc6). The superiority of bacterial laccase over fungal laccase was elucidated from the perspective of crystal structure. Besides, a variety of technical means were used to verify the success of surface-display. pGSA-CotA surface-displayed bacteria exhibited good tolerance to high temperature, pH, and various heavy metals. Importantly, surface-displayed bacteria showed faster degradation efficiency and better treatment effects than the intracellular expression bacteria in tetracycline degradation. This implies that surface display technology has greater potential for laccase-mediated environmental remediation. Due to the adverse impacts of tetracycline on soil enzyme activity and microorganisms, our study found that pGSA-CotA surface-displayed bacteria can alleviate tetracycline stress in soil and partially activate the soil, thereby increasing soil enzyme activity and certain nitrogen cycling genes.

Genetically surface-modified Escherichia coli outer membrane vesicles targeting MUC1 antigen in cancer cells.

Outer membrane vesicles (OMVs), non-replicating spherical liposomes derived from Gram-negative bacteria, are a promising vaccine platform and multifunctional delivery systems. Their ability to be modified via genetic engineering for the incorporation and display of heterologous proteins enhances their functionality. In this study, we demonstrated a bio-ligation approach to display single-chain variable fragments (scFv) on the OMV surface using the SpyTag/SpyCatcher system. SpyTag-fused scFv, expressed by mammalian cells, bound to OMVs with SpyCatcher-fused Lpp'OmpA after a simple incubation. Biophysical analysis indicated that the conjugated OMVs maintained their physicochemical properties. We used an scFv targeting mucin 1 protein (MUC1) for specific cell targeting. Confocal microscopy revealed that conjugated OMVs specifically bound to and were internalized by MUC1-presenting cells, but not by MUC1-deficient cells. In conclusion, this rapid and efficient bio-ligation system facilitates the display of functional scFv on OMV surfaces, offering a promising approach for targeted delivery to MUC1-expressing cancer cells.

Display of Bacterial Exochitanase on Bacillus subtilis Spores Improved Enzyme Stability and Recyclability.

Chitin is the second most prevalent polysaccharide found in nature, following cellulose. Amino-oligosaccharides, the byproducts of chitin degradation, exhibit favorable biological properties and potential for various uses. Chitinases play a crucial function in the breakdown of chitin, and their exceptionally effective production has garnered significant interest. Here, in this study, the exochitinase PbChiA, obtained from Paenibacillus barengoltzii, was recombinantly produced and immobilized using the CotG surface protein of Bacillus subtilis WB800N. The resulting strain Bacillus subtilis WB800N pHS-CotG-Chi exhibited exceptional heat stability and efficacy across various pH levels. The chitinolytic activity of the enzyme, which had been isolated and immobilized on the spore surface, was measured to be approximately 16.06 U/mL. Including Ni2+, Zn+2, and K+, and EDTA at various concentration levels in the reaction system, has significantly enhanced the activity of the immobilized enzyme. The immobilized exochitinase demonstrated a notable rate of recycling, as the recombinant spores sustained a relative enzyme activity of more than 70% after three cycles and 62.7% after four cycles. These findings established a basis for additional investigation into the role and practical use of the immobilized bacterial exochitinase in industry.

Targeted aggregation of PETase towards surface of Stenotrophomonas pavanii for degradation of PET microplastics.

Polyethylene terephthalate (PET) is one of the most widely used plastics, but its fragmentation into microplastics poses significant environmental challenges. The recycling of PET microplastics is hindered by their low solubility and widespread dispersion in the environment, making microbial in-situ degradation a promising solution. However, existing PET-degrading strains exhibited the limited effectiveness, primarily due to the diffusion of secreted hydrolases away from the PET surface. In this study, Stenotrophomonas pavanii JWG-G1 was engineered to achieve the targeted aggregation of PET hydrolase PETase on the cell surface by fusing it with an endogenous anchor protein. This approach aims to maximise the local concentration of PETase around PET, thereby increasing the overall rate of PET degradation. The PETase surface-aggregated system, S. pavanii/PaL-PETase, demonstrated the highest degradation efficiency, achieving 63.3 % degradation of low-crystallinity PET (lcPET) and 27.3 % degradation of high-crystallinity PET bottles (hcPET) at 30 °C. This represents the highest degradation rate reported for a displayed whole-cell system at ambient temperature. Furthermore, this system exhibited broad-spectrum degradation activity against various polyesters. These findings suggest that this system offers a promising, eco-friendly solution to PET and other polyester pollution, with potential implications for environmental bioremediation strategies.

A platform for the early selection of non-competitive antibody-fragments from yeast surface display libraries.

In this work, we report the development of a platform for the early selection of non-competitive antibody-fragments against cell surface receptors that do not compete for binding of their natural ligand. For the isolation of such subtype of blocking antibody-fragments, we applied special fluorescence-activated cell sorting strategies for antibody fragments isolation from yeast surface display libraries. Given that most of the monoclonal antibodies approved on the market are blocking ligand-receptor interactions often leading to resistance and/or side effects, targeting allosteric sites represents a promising mechanism of action to open new avenues for treatment. To directly identify these antibody-fragments during library screening, we employed immune libraries targeting the epidermal growth factor receptor as proof of concept. Incorporating a labeled orthosteric ligand during library sorting enables the early selection of non-competitive binders and introduces an additional criterion to refine the selection of candidates exhibiting noteworthy properties. Furthermore, after sequencing, more candidates were identified compared to classical sorting based solely on target binding. Hence, this platform can significantly improve the drug discovery process by the early selection of more candidates with desired properties.

Engineering Adhesion of the Probiotic Strain Escherichia coli Nissle to the Fungal Pathogen Candida albicans.

Engineering live biotherapeutic products against fungal pathogens such as Candida albicans has been suggested as a means to tackle the increasing threat of fungal infections and the development of resistance to classical antifungal treatments. One important challenge in the design of live therapeutics is to control their localization inside the human body. The specific binding capability to target organisms or tissues would greatly increase their effectiveness by increasing the local concentration of effector molecules at the site of infection. In this study, we utilized surface display of carbohydrate binding domains to enable the probiotic E. coli Nissle 1917 to adhere specifically to the pathogenic yeast Candida albicans. Binding was quantified using a newly developed method based on the automated analysis of microscopic images. In addition to a rationally selected chitin binding domain, a synthetic peptide of identical length but distinct sequence also conferred binding. Efficient binding was specific to fungal hyphae, the invasive form of C. albicans, while the yeast form, as well as abiotic cellulose and PET particles, was only weakly recognized.

Yeast Surface-Displayed Quenchbody as a Novel Whole-Cell Biosensor for One-Step Detection of Influenza A (H1N1) Virus.

Timely surveillance of airborne pathogens is essential to preventing the spread of infectious diseases and safeguard human health. Methods for sensitive, efficient, and cost-effective detection of airborne viruses are needed. With advances in synthetic biology, whole-cell biosensors have emerged as promising platforms for environmental monitoring and medical diagnostics. However, the current design paradigm of whole-cell biosensors is mostly based on intracellular detection of analytes that can transport across the cell membrane, which presents a critical challenge for viral pathogens and large biomolecules. To address this challenge, we developed a new type of whole-cell biosensor by expressing and displaying VHH-based quenchbody (Q-body) on the surface of the yeast Saccharomyces cerevisiae for simple one-step detection of influenza A (H1N1) virus. Seventeen VHH antibody fragments targeting the hemagglutinin protein H1N1-HA were displayed on the yeast cells and screened for the H1N1-HA binding affinity. The functionally displayed VHHs were selected to create surface-displayed Q-body biosensors. The surface-displayed Q-body exhibiting the highest quenching and dequenching efficiency was identified. The biosensor quantitatively detected H1N1-HA in a range from 0.5 to 16 µg/mL, with a half-maximal concentration of 2.60 µg/mL. The biosensor exhibited high specificity for H1N1-HA over other hemagglutinin proteins from various influenza A virus subtypes. Moreover, the biosensor succeeded in detecting the H1N1 virus at concentrations from 2.4 × 104 to 1.5 × 107 PFU/mL. The results from this study demonstrated a new whole-cell biosensor design that circumvents the need for transport of analytes into biosensor cells, enabling efficient detection of the target virus particles.

Yeast surface display technology: Mechanisms, applications, and perspectives.

Microbial cell surface display technology, which relies on genetically fusing heterologous target proteins to the cell wall through fusion with cell wall anchor proteins, has emerged as a promising and powerful method with diverse applications in biotechnology and biomedicine. Compared to classical intracellular or extracellular expression (secretion) systems, the cell surface display strategy stands out by eliminating the necessity for enzyme purification, overcoming substrate transport limitations, and demonstrating enhanced activity, stability, and selectivity. Unlike phage or bacterial surface display, the yeast surface display (YSD) system offers distinct advantages, including its large cell size, ease of culture and genetic manipulation, the use of generally regarded as safe (GRAS) host cell, the ability to ensure correct folding of complex eukaryotic proteins, and the potential for post-translational modifications. To date, YSD systems have found widespread applications in protein engineering, waste biorefineries, bioremediation, and the production of biocatalysts and biosensors. This review focuses on detailing various strategies and mechanisms for constructing YSD systems, providing a comprehensive overview of both fundamental principles and practical applications. Finally, the review outlines future perspectives for developing novel forms of YSD systems and explores potential applications in diverse fields.

On-Demand Vaccine Production via Dock-and-Display of Biotinylated Antigens on Bacterial Extracellular Vesicles.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs.

Immune Responses Induced by a Recombinant Lactiplantibacillus plantarum Surface-Displaying the gD Protein of Pseudorabies Virus.

Pseudorabies virus (PRV) is one of the herpes viruses that can infect a wide range of animals including pigs, cattle, sheep, mice, and wild animals. PRV is a neurotropic alphaherpesvirus capable of infecting a variety of mammals. There is a rising interest in the targeted application of probiotic bacteria to prevent viral diseases, including PRV. In this study, the surface expression of enhanced green fluorescent protein (EGFP) on recombinant Lactiplantibacillus plantarum NC8 (rNC8) through the LP3065 LPxTG motif of Lactobacillus plantarum WCFS1 was generated. The surface expression was observed through confocal microscopy. Dendritic cell targeting peptides (DCpep) were also fused with LPxTG that help to bind with mouse DCs. The PRV-gD was cloned in LP3065 LPxTG, resulting in the generation of rNC8-LP3065-gD. Inactivated rNC8-LP3065-gD was administered intravenously in mice on days 1 and 7 at a dose of 200 µL (109 CFU/mouse) for monitoring immunogenicity. Subsequently, a challenge dose of PRV TJ (104 TCID50) was administered intramuscularly at 14 days post-immunization. The survival rate of the immunized mice reached 80% (4/5) with no significant signs of illness. A significant rise in anti-gD antibodies was detected in the immunized mice by ELISA. Quantitative PCR (qPCR) results showed decreased viral loading in different body tissues. Flow cytometry of lymphocytes derived from mice spleen indicated an increase in CD3+CD4+ T cells, but CD3+CD8+ T cells were not detected. Moreover, it offers a model to delineate immune correlates with rNC8-induced immunity against swine viral diseases.

T cell receptor-directed antibody-drug conjugates for the treatment of T cell-derived cancers.

T cell-derived cancers are hallmarked by heterogeneity, aggressiveness, and poor clinical outcomes. Available targeted therapies are severely limited due to a lack of target antigens that allow discrimination of malignant from healthy T cells. Here, we report a novel approach for the treatment of T cell diseases based on targeting the clonally rearranged T cell receptor displayed by the cancerous T cell population. As a proof of concept, we identified an antibody with unique specificity toward a distinct T cell receptor (TCR) and developed antibody-drug conjugates, precisely recognizing and eliminating target T cells while preserving overall T cell repertoire integrity and cellular immunity. Our anti-TCR antibody-drug conjugates demonstrated effective receptor-mediated cell internalization, associated with induction of cancer cell death with strong signs of apoptosis. Furthermore, cell proliferation-inhibiting bystander effects observed on target-negative cells may contribute to the molecules' anti-tumor properties precluding potential tumor escape mechanisms. To our knowledge, this represents the first anti-TCR antibody-drug conjugate designed as custom-tailored immunotherapy for T cell-driven pathologies.

Improved Prediction of Stabilizing Mutations in Proteins by Incorporation of Mutational Effects on Ligand Binding.

While many computational methods accurately predict destabilizing mutations, identifying stabilizing mutations has remained a challenge, because of their relative rarity. We tested ΔΔG0 predictions from computational predictors such as Rosetta, ThermoMPNN, RaSP, and DeepDDG, using 82 mutants of the bacterial toxin CcdB as a test case. On this dataset, the best computational predictor is ThermoMPNN, which identifies stabilizing mutations with a precision of 68%. However, the average increase in Tm for these predicted mutations was only 1°C for CcdB, and predictions were poorer for a more challenging target, influenza neuraminidase. Using data from multiple previously described yeast surface display libraries and in vitro thermal stability measurements, we trained logistic regression models to identify stabilizing mutations with a precision of 90% and an average increase in Tm of 3°C for CcdB. When such libraries contain a population of mutants with significantly enhanced binding relative to the corresponding wild type, there is no benefit in using computational predictors. It is then possible to predict stabilizing mutations without any training, simply by examining the distribution of mutational binding scores. This avoids laborious steps of in vitro expression, purification, and stability characterization. When this is not the case, combining data from computational predictors with high-throughput experimental binding data enhances the prediction of stabilizing mutations. However, this requires training on stability data measured in vitro with known stabilized mutants. It is thus feasible to predict stabilizing mutations rapidly and accurately for any system of interest that can be subjected to a binding selection or screen.

Measuring the capacity of yeast for surface display of cell wall-anchored protein isoforms by using ß-lactamase as a reporter enzyme.

Yeast surface display is a promising biotechnological tool that uses genetically modified yeast cell wall proteins as anchors for enzymes of interest, thereby transforming yeast cell wall into a living catalytic material. Here, we present a comprehensive protocol for quantifying surface-displayed ß-lactamase on the cell wall of model yeast Saccharomyces cerevisiae. We use ß-lactamase as a reporter enzyme, which we tagged to be anchored to the cell wall closer to its N or C terminus, through the portion of the Pir2 or Ccw12 cell wall proteins, respectively. The catalytic activity of surface-displayed ß-lactamase is assessed by its ability to hydrolyze nitrocefin, which produces a colorimetric change that is quantitatively measured by spectrophotometric analysis at 482 nm. This system enables precise quantification of the potential of S. cerevisiae strains for surface display, continuous real-time monitoring of enzyme activity, and facilitates the study of enzyme kinetics and interactions with inhibitors within the cell's native environment. In addition, the system provides a platform for high-throughput screening of potential ß-lactamase inhibitors and can be adapted for the visualization of other enzymes, making it a versatile tool for drug discovery and bioprocess development.

Bacterial surface display of PETase mutants and MHETase for an efficient dual-enzyme cascade catalysis.

Biological degradation of PET plastic holds great potential for plastic recycling. However, the high costs associated with preparing free enzymes for degrading PET make it unfeasible for industrial applications. Hence, we developed various cell catalysts by surface-displaying PETase mutants and MHETase using autotransporters in E. coli and P. putida. The efficiency of surface display was enhanced through modifying the host, co-expressing molecular chaperones, and evoluting the autotransporter. In strain EC9F, PET degradation rate was boosted to 3.85 mM/d, 51-fold and 23-fold increase compared to free enzyme and initial strain ED1, respectively. The reusability of cell catalyst EC9F was demonstrated with over 38 % and 30 % of its initial activity retained after 22 cycles of BHET degradation and 3 cycles of PET degradation. The highest reported PET degradation rate of 4.95 mM/d was achieved by the dual-enzyme cascade catalytic system EC9F+EM2+R, a mixture of cell catalyst EC9F and EM2 with surfactant rhamnolipid.