Disentangling the Web: An Interdisciplinary Review on the Potential and Feasibility of Spider Silk Bioproduction.

The remarkable material properties of spider silk, such as its high toughness and tensile strength combined with its low density, make it a highly sought-after material with myriad applications. In addition, the biological nature of spider silk makes it a promising, potentially sustainable alternative to many toxic or petrochemical-derived materials. Therefore, interest in the heterologous production of spider silk proteins has greatly increased over the past few decades, making recombinant spider silk an important frontier in biomanufacturing. This has resulted in a diversity of potential host organisms, a large space for sequence design, and a variety of downstream processing techniques and product applications for spider silk production. Here, we highlight advances in each of these technical aspects as well as white spaces therein, still ripe for further investigation and discovery. Additionally, industry landscaping, patent analyses, and interviews with Key Opinion Leaders help define both the research and industry landscapes. In particular, we found that though textiles dominated the early products proposed by companies, the versatile nature of spider silk has opened up possibilities in other industries, such as high-performance materials in automotive applications or biomedical therapies. While continuing enthusiasm has imbued scientists and investors alike, many technical and business considerations still remain unsolved before spider silk can be democratized as a high-performance product. We provide insights and strategies for overcoming these initial hurdles, and we highlight the importance of collaboration between academia, industry, and policy makers. Linking technical considerations to business and market entry strategies highlights the importance of a holistic approach for the effective scale-up and commercial viability of spider silk bioproduction.

Spider Silk-Inspired Hyaluronic Acid-Based Hydrogels with Superior Self-Healing Capability and Enhanced Strength.

Hyaluronic acid hydrogels are promising materials for diverse applications, yet their potential is hampered by limitations such as low self-healing efficiency and insufficient mechanical strength. Inspired by the heterogeneous structures of spider silk, we introduce a novel dual dynamically crosslinked network hydrogel. This hydrogel comprises an acylhydrazone-crosslinked network, utilizing aldehyde hyaluronic acid (AHA) and 3,3'-dithiobis (propionohydrazide) (DTP) as a first network, and a secondary network formed by hydrogen bonds-crosslinked network between tannic acid (TA) and silk fibroin (SF) with ß-sheet formation. The hydrogel exhibits exceptional self-healing ability due to the dynamic and reversible nature of Schiff base bonds, disulfide bonds, and hydrogen bonds, achieving complete healing within 5 minutes. Additionally, the spider silk-inspired heterogeneous structures enhance mechanical properties. Furthermore, the incorporation of TA provides enhanced adhesion, as well as remarkable antibacterial and antioxidant properties. This innovative hyaluronic acid-based hydrogel, inspired by spider silk, offers a promising avenue to fortify both the mechanical strength and self-healing capabilities of hydrogels, thus expanding opportunities for applications in tissue engineering and biomedicine.

Hierarchical looping results in extreme extensibility of silk fibre composites produced by Southern house spiders (Kukulcania hibernalis).

Spider silk is a tough and versatile biological material combining high tensile strength and extensibility through nanocomposite structure and its nonlinear elastic behaviour. Notably, spiders rarely use single silk fibres in isolation, but instead process them into more complex composites, such as silk fibre bundles, sheets and anchorages, involving a combination of spinneret, leg and body movements. While the material properties of single silk fibres have been extensively studied, the mechanical properties of silk composites and meta-structures are poorly understood and exhibit a hereto largely untapped potential for the bio-inspired design of novel fabrics with outstanding mechanical properties. In this study, we report on the tensile mechanics of the adhesive capture threads of the Southern house spider (Kukulcania hibernalis), which exhibit extreme extensibility, surpassing that of the viscid capture threads of orb weavers by up to tenfold. By combining high-resolution mechanical testing, microscopy and in silico experiments based on a hierarchical modified version of the Fibre Bundle Model, we demonstrate that extreme extensibility is based on a hierarchical loops-on-loops structure combining linear and coiled elements. The stepwise unravelling of the loops leads to the repeated fracture of the connected linear fibres, delaying terminal failure and enhancing energy absorption. This principle could be used to achieve tailored fabrics and materials that are able to sustain high deformation without failure.

From fibres to adhesives: evolution of spider capture threads from web anchors by radical changes in silk gland function.

Spider webs that serve as snares are one of the most fascinating and abundant type of animal architectures. In many cases they include an adhesive coating of silk lines-so-called viscid silk-for prey capture. The evolutionary switch from silk secretions forming solid fibres to soft aqueous adhesives remains an open question in the understanding of spider silk evolution. Here we functionally and chemically characterized the secretions of two types of silk glands and their behavioural use in the cellar spider, Pholcus phalangioides. Both being derived from the same ancestral gland type that produces fibres with a solidifying glue coat, the two types produce respectively a quickly solidifying glue applied in thread anchorages and prey wraps, or a permanently tacky glue deployed in snares. We found that the latter is characterized by a high concentration of organic salts and reduced spidroin content, showing up a possible pathway for the evolution of viscid properties by hygroscopic-salt-mediated hydration of solidifying adhesives. Understanding the underlying molecular basis for such radical switches in material properties not only helps to better understand the evolutionary origins and versatility of ecologically impactful spider web architectures, but also informs the bioengineering of spider silk-based products with tailored properties.

Nanoassembly of spider silk protein mediated by intrinsically disordered regions.

Spider silk is the self-assembling product of silk proteins each containing multiple repeating units. Each repeating unit is entirely intrinsically disordered or contains a small disordered domain. The role of the disordered domain/unit in conferring silk protein storage and self-assembly is not fully understood yet. Here, we used biophysical and biochemical techniques to investigate the self-assembly of a miniature version of a minor ampullate spidroin (denoted as miniMiSp). miniMiSp consists of two identical intrinsically disordered domains, one folded repetitive domain, and two folded terminal domains. Our data indicated that miniMiSp self-assembles into oligomers and further into liquid droplets. The oligomerization is attributed to the aggregation-prone property of both the disordered domains and the folded repetitive domain. Our results support the model of micellar structure for silk proteins at high protein concentrations. The disordered domain is indispensable for liquid droplet formation via liquid-liquid phase separation, and tyrosine residues located in the disordered domain make dominant contributions to stability of the liquid droplets. As the same tyrosine residues are also critical to fibrillation, the liquid droplets are likely an intermediate state between the solution state and the fiber state. Additionally, the terminal domains contribute to the pH- and salt-dependent self-assembly properties.

Interfacial interactions between spider silk protein and cellulose studied by molecular dynamics simulation.

CONTEXT: Due to their excellent biocompatibility and degradability, cellulose/spider silk protein composites hold a significant value in biomedical applications such as tissue engineering, drug delivery, and medical dressings. The interfacial interactions between cellulose and spider silk protein affect the properties of the composite. Therefore, it is important to understand the interfacial interactions between spider silk protein and cellulose to guide the design and optimization of composites. The study of the adsorption of protein on specific surfaces of cellulose crystal can be very complex using experimental methods. Molecular dynamics simulations allow the exploration of various physical and chemical changes at the atomic level of the material and enable an atomic description of the interactions between cellulose crystal planes and spider silk protein. In this study, molecular dynamics simulations were employed to investigate the interfacial interactions between spider silk protein (NTD) and cellulose surfaces. Findings of RMSD, RMSF, and secondary structure showed that the structure of NTD proteins remained unchanged during the adsorption process. Cellulose contact numbers and hydrogen bonding trends on different crystalline surfaces suggest that van der Waals forces and hydrogen bonding interactions drive the binding of proteins to cellulose. These findings reveal the interaction between cellulose and protein at the molecular level and provide theoretical guidance for the design and synthesis of cellulose/spider silk protein composites. METHODS: MD simulations were all performed using the GROMACS-5.1 software package and run with CHARMM36 carbohydrate force field. Molecular dynamics simulations were performed for 500 ns for the simulated system.

Special Prey, Special Glue: NMR Spectroscopy on Aggregate Glue Components of Moth-Specialist Spiders, Cyrtarachninae.

Orb-weaver spiders produce upwards of seven different types of silk, each with unique material properties. We focus on the adhesive within orb-weaving spider webs, aggregate glue silk. These droplets are composed of three main components: water, glycoproteins, and a wide range of low molecular mass compounds (LMMCs). These LMMCs are known to play a crucial role in maintaining the material properties of the glycoproteins, aid in water absorption from the environment, and increase surface adhesion. Orb-weavers within the Cyrtarachninae subfamily are moth specialists and have evolved glue droplets with novel material properties. This study investigated the biochemical composition and diversity of the LMMCs present in the aggregate glue of eight moth-specialist species and compared them with five generalist orb-weavers using nuclear magnetic resonance (NMR) spectroscopy. We hypothesized that the novel drying ability of moth-specialist glue was accompanied by novel LMMCs and lower overall percentages by silk weight of LMMCs. We measured no difference in LMMC weight by the type of prey specialization, but observed novel compositions in the glue of all eight moth-catching species. Further, we quantified the presence of a previously reported but unidentified compound that appears in the glue of all moth specialists. These silks can provide insight into the functions of bioadhesives and inform our own synthetic adhesives.

Modulating Polyalanine Motifs of Synthetic Spidroin for Controllable Preassembly and Strong Fiber Formation.

Spider dragline (major ampullate) silk is one of the toughest known fibers in nature and exhibits an excellent combination of high tensile strength and elasticity. Increasing evidence has indicated that preassembly plays a crucial role in facilitating the proper assembly of silk fibers by bridging the mesoscale gap between spidroin molecules and the final strong fibers. However, it remains challenging to control the preassembly of spidroins and investigate its influence on fiber structural and mechanical properties. In this study, we explored to bridge this gap by modulating the polyalanine (polyA) motifs in repetitive region of spidroins to tune their preassemblies in aqueous dope solutions. Three biomimetic silk proteins with varying numbers of alanine residues in polyA motif and comparable molecular weights were designed and biosynthesized, termed as N16C-5A, N15C-8A, and N13C-12A, respectively. It was found that all three proteins could form nanofibril assemblies in the concentrated aqueous dopes, but the size and structural stability of the fibrils were distinct from each other. The silk protein N15C-8A with 8 alanine residues in polyA motif allowed for the formation of stable nanofibril assemblies with a length of approximately 200 nm, which were not prone to disassemble or aggregate as that of N16C-5A and N13C-12A. More interestingly, the stable fibril assembly of N15C-8A enabled spinning of simultaneously strong (623.3 MPa) and tough (107.1 MJ m-3) synthetic fibers with fine molecular orientation and close interface packing of fibril bundles. This work highlights that modulation of polyA motifs is a feasible way to tune the morphology and stability of the spidroin preassemblies in dope solutions, thus controlling the structural and mechanical properties of the resulting fibers.

Custom-designed, mass silk production in genetically engineered silkworms.

Genetically engineered silkworms have been widely used to obtain silk with modified characteristics especially by introducing spider silk genes. However, these attempts are still challenging due to limitations in transformation strategies and difficulties in integration of the large DNA fragments. Here, we describe three different transformation strategies in genetically engineered silkworms, including transcription-activator-like effector nuclease (TALEN)-mediated fibroin light chain (FibL) fusion (BmFibL-F), TALEN-mediated FibH replacement (BmFibH-R), and transposon-mediated genetic transformation with the silk gland-specific fibroin heavy chain (FibH) promoter (BmFibH-T). As the result, the yields of exogenous silk proteins, a 160 kDa major ampullate spidroin 2 (MaSp2) from the orb-weaving spider Nephila clavipes and a 226 kDa fibroin heavy chain protein (EvFibH) from the bagworm Eumeta variegate, reach 51.02 and 64.13% in BmFibH-R transformed cocoon shells, respectively. Moreover, the presence of MaSp2 or EvFibH significantly enhances the toughness of genetically engineered silk fibers by ∼86% in BmFibH-T and ∼80% in BmFibH-R silkworms, respectively. Structural analysis reveals a substantial ∼40% increase in fiber crystallinity, primarily attributed to the presence of unique polyalanines in the repetitive sequences of MaSp2 or EvFibH. In addition, RNA-seq analysis reveals that BmFibH-R system only causes minor impact on the expression of endogenous genes. Our study thus provides insights into developing custom-designed silk production using the genetically engineered silkworm as the bioreactor.

Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco.

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2-3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.

In vitro and in vivo investigation of Argiope bruennichi spider silk-based novel biomaterial for medical use.

As a natural and biocompatible material with high strength and flexibility, spider silk is frequently used in biomedical studies. In this study, the availability of Argiope bruennichi spider silk as a surgical suture material was investigated. The effects of spider silk-based and commercial sutures, with and without Aloe vera coating, on wound healing were evaluated by a rat dorsal skin flap model, postoperatively (7th and 14th days). Biochemical, hematological, histological, immunohistochemical, small angle x-ray scattering (SAXS) analyses and mechanical tests were performed. A. bruennichi silk did not show any cytotoxic effect on the L929 cell line according to MTT and LDH assays, in vitro. The silk materials did not cause any allergic reaction, infection, or systemic effect in rats according to hematological and biochemical analyses. A. bruennichi spider silk group showed a similar healing response to commercial sutures. SAXS analysis showed that the 14th-day applications of A. bruennichi spider silk and A. vera coated commercial suture groups have comparable structural results with control group. In conclusion, A. bruennichi spider silk is biocompatible in line with the parameters examined and shows a healing response similar to the commercial sutures commonly used in the skin.

Quantitative Shotgun Proteomic Analysis of Bacteria after Overexpression of Recombinant Spider Miniature Spidroin, MaSp1.

Spider silk has extraordinary mechanical properties, displaying high tensile strength, elasticity, and toughness. Given the high performance of natural fibers, one of the long-term goals of the silk community is to manufacture large-scale synthetic spider silk. This process requires vast quantities of recombinant proteins for wet-spinning applications. Attempts to synthesize large amounts of native size recombinant spidroins in diverse cell types have been unsuccessful. In these studies, we design and express recombinant miniature black widow MaSp1 spidroins in bacteria that incorporate the N-terminal and C-terminal domain (NTD and CTD), along with varying numbers of codon-optimized internal block repeats. Following spidroin overexpression, we perform quantitative analysis of the bacterial proteome to identify proteins associated with spidroin synthesis. Liquid chromatography with tandem mass spectrometry (LC MS/MS) reveals a list of molecular targets that are differentially expressed after enforced mini-spidroin production. This list included proteins involved in energy management, proteostasis, translation, cell wall biosynthesis, and oxidative stress. Taken together, the purpose of this study was to identify genes within the genome of Escherichia coli for molecular targeting to overcome bottlenecks that throttle spidroin overexpression in microorganisms.

Spider's Silk as a Potential Source of Antibiotics: An Integrative Review.

Spiders produce webs, which are still a largely unexplored source of antibacterial compounds, although the reports of its application in the medical field. Therefore, this study aims to present an integrative review of the antibacterial activity of spider webs. The research was conducted using Google Scholar, Scielo, Web of Science, PubMed, ScienceDirect, Medline EBSCO, LILACS, and Embase. The inclusion criteria were original articles written in English that studied the antibiotic properties of the web or isolated compounds tested. The studies were compared according to the spider species studied, the type of web, treatment of the sample, type of antimicrobial test, and the results obtained. Nine hundred and seventy-three publications were found, and after applying the inclusion and exclusion criteria, sixteen articles were selected. Bacterial inhibition was found in seven studies against various species of bacteria such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella Typhi, Bacillus megaterium, Listeria monocytogenes, Acinetobacter baumannii, Streptococcus pneumoniae, Pasteurella multocida, and Bacillus subtilis. Additionally, there was no apparent relationship between the proximity of the spider species evaluated in the studies and the presence or absence of activity. Methodological problems detected may affected the reproducibility and reliability of the results in some studies, such as the lack of description of the web or microorganism strain, as well as the absence of adequate controls and treatments to sterilize the sample. Spider webs can be a valuable source of antibiotics; however, more studies are needed to confirm the real activity of the web or components involved.

Biocomposite Materials Derived from Andropogon halepensis: Eco-Design and Biophysical Evaluation.

This research work presents a "green" strategy of weed valorization for developing silver nanoparticles (AgNPs) with promising interesting applications. Two types of AgNPs were phyto-synthesized using an aqueous leaf extract of the weed Andropogon halepensis L. Phyto-manufacturing of AgNPs was achieved by two bio-reactions, in which the volume ratio of (phyto-extract)/(silver salt solution) was varied. The size and physical stability of Andropogon-AgNPs were evaluated by means of DLS and zeta potential measurements, respectively. The phyto-developed nanoparticles presented good free radicals-scavenging properties (investigated via a chemiluminescence technique) and also urease inhibitory activity (evaluated using the conductometric method). Andropogon-AgNPs could be promising candidates for various bio-applications, such as acting as an antioxidant coating for the development of multifunctional materials. Thus, the Andropogon-derived samples were used to treat spider silk from the spider Pholcus phalangioides, and then, the obtained "green" materials were characterized by spectral (UV-Vis absorption, FTIR ATR, and EDX) and morphological (SEM) analyses. These results could be exploited to design novel bioactive materials with applications in the biomedical field.

[Primary Structure Characterization and Biosynthesis of Spider Silk Proteins for Multifunctional Biomaterials].

Spider silk is a natural fiber known as "biosteel" with the strongest composite performance, such as high tensile strength and toughness. It is also equipped with excellent biocompatibility and shape memory ability, thus shows great potential in many fields such as biomedicine and tissue engineering. Spider silk is composed of macromolecular spidroin with rich structural diversity. The characteristics of the primary structure of natural spidroin, such as the high repeatability of amino acids in the core repetitive region, the high content of specific amino acids, the large molecular weight, and the high GC content of the spidroin gene, have brought great difficulties in heterologous expression. This review discusses focuses on the relationship between the featured motifs of the microcrystalline region in the repetitive unit of spidroin and its structure, as well as the spinning performance and the heterologous expression. The optimization design for the sequence of spidroin combined with heterologous expression strategy has greatly promoted the development of the biosynthesis of spider silk proteins. This review may facilitate the rational design and efficient synthesis of recombinant spidroin.

Influence of Spider Silk Protein Structure on Mechanical and Biological Properties for Energetic Material Detection.

Spider silk protein, renowned for its excellent mechanical properties, biodegradability, chemical stability, and low immune and inflammatory response activation, consists of a core domain with a repeat sequence and non-repeating sequences at the N-terminal and C-terminal. In this review, we focus on the relationship between the silk structure and its mechanical properties, exploring the potential applications of spider silk materials in the detection of energetic materials.

Review of Spider Silk Applications in Biomedical and Tissue Engineering.

This review will present the latest research related to the production and application of spider silk and silk-based materials in reconstructive and regenerative medicine and tissue engineering, with a focus on musculoskeletal tissues, and including skin regeneration and tissue repair of bone and cartilage, ligaments, muscle tissue, peripheral nerves, and artificial blood vessels. Natural spider silk synthesis is reviewed, and the further recombinant production of spider silk proteins. Research insights into possible spider silk structures, like fibers (1D), coatings (2D), and 3D constructs, including porous structures, hydrogels, and organ-on-chip designs, have been reviewed considering a design of bioactive materials for smart medical implants and drug delivery systems. Silk is one of the toughest natural materials, with high strain at failure and mechanical strength. Novel biomaterials with silk fibroin can mimic the tissue structure and promote regeneration and new tissue growth. Silk proteins are important in designing tissue-on-chip or organ-on-chip technologies and micro devices for the precise engineering of artificial tissues and organs, disease modeling, and the further selection of adequate medical treatments. Recent research indicates that silk (films, hydrogels, capsules, or liposomes coated with silk proteins) has the potential to provide controlled drug release at the target destination. However, even with clear advantages, there are still challenges that need further research, including clinical trials.

Unraveling the Mechanical Property Decrease of Electrospun Spider Silk: A Molecular Dynamics Simulation Study.

This study investigated the impact of electric fields on Nephila clavipes spider silk using molecular dynamics modeling. Electric fields with varying amplitudes and directions were observed to disrupt the ß sheet structure of spider silk and reduce its mechanical properties. However, a notable exception was observed when a 0.1 V/nm electric field was applied in the antiparallel direction, resulting in improvements in Young's modulus and ultimate tensile strength. The antiparallel direction was observed to be particularly sensitive to electric fields, causing disruptions in beta sheets and hydrogen bonds, which significantly influence the mechanical properties. This study demonstrates that spider silk maintains its structural integrity at 0.1 V/nm. Possibly, lowering the power levels of typical electrospinning machines can prevent secondary structural disruption. These findings provide valuable insights for enhancing silk fiber production and applications using natural silk proteins while shedding light on the impact of electric fields on other silk proteins. Finally, this study opens up possibilities for optimizing electrospinning processes to enhance performance in various silk electrospinning applications.

Fabrication and biomechanical characterization of a spider silk reinforced fibrin-based vascular prosthesis.

With fibrin-based vascular prostheses, vascular tissue engineering offers a promising approach for the fabrication of biologically active regenerative vascular grafts. As a potentially autologous biomaterial, fibrin exhibits excellent hemo- and biocompatibility. However, the major problem in the use of fibrin constructs in vascular tissue engineering, which has so far prevented their widespread clinical application, is the insufficient biomechanical stability of unprocessed fibrin matrices. In this proof-of-concept study, we investigated to what extent the addition of a spider silk network into the wall structure of fibrin-based vascular prostheses leads to an increase in biomechanical stability and an improvement in the biomimetic elastic behavior of the grafts. For the fabrication of hybrid prostheses composed of fibrin and spider silk, a statically cast tubular fibrin matrix was surrounded with an envelope layer of Trichonephila edulis silk using a custom built coiling machine. The fibrin matrix was then compacted and pressed into the spider silk network by transluminal balloon compression. This manufacturing process resulted in a hybrid prosthesis with a luminal diameter of 4 mm. Biomechanical characterization revealed a significant increase in biomechanical stability of spider silk reinforced grafts compared to exclusively compacted fibrin segments with a mean burst pressure of 362 ± 74 mmHg vs. 213 ± 14 mmHg (p < 0.05). Dynamic elastic behavior of the spider silk reinforced grafts was similar to native arteries. In addition, the coiling with spider silk allowed a significant increase in suture retention strength and resistance to external compression without compromising the endothelialization capacity of the grafts. Thus, spider silk reinforcement using the abluminal coiling technique represents an efficient and reproducible technique to optimize the biomechanical behavior of small-diameter fibrin-based vascular grafts.

Superfast Gelation of Spider Silk-Based Artificial Silk Protein.

Spider silk proteins (spidroins) have garnered attention in biomaterials research due to their ability to self-assemble into hydrogels. However, reported spidroin hydrogels require high protein concentration and prolonged gelation time. Our study engineered an artificial spidroin that exhibits unprecedented rapid self-assembly into hydrogels at physiologically relevant conditions, achieving gelation at a low concentration of 6 mg/mL at 37 °C without external additives. Remarkably, at a 30 mg/mL concentration, our engineered protein forms hydrogels within 30 s, a feature we termed "superfast gelation". This rapid formation is modulated by ions, pH, and temperature, offering versatility in biomedical applications. The hydrogel's capacity to encapsulate proteins and support E. coli growth while inducing RFP expression provides a novel platform for drug delivery and bioengineering applications. Our findings introduce a superfast, highly adaptable, and cytocompatible hydrogel that self-assembles under mild conditions, underscoring the practical implication of rapid gelation in biomedical research and clinical applications.