Mass spider silk production through targeted gene replacement in Bombyx mori.

Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, Bombyx mori, has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in B. mori by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) in the spider Nephila clavipes We successfully replaced the ∼16-kb endogenous FibH gene with a 1.6-kb MaSp1 gene fused with a 1.1-kb partial FibH sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted MaSp1 integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.

Hierarchical spidroin micellar nanoparticles as the fundamental precursors of spider silks.

Many natural silks produced by spiders and insects are unique materials in their exceptional toughness and tensile strength, while being lightweight and biodegradable-properties that are currently unparalleled in synthetic materials. Myriad approaches have been attempted to prepare artificial silks from recombinant spider silk spidroins but have each failed to achieve the advantageous properties of the natural material. This is because of an incomplete understanding of the in vivo spidroin-to-fiber spinning process and, particularly, because of a lack of knowledge of the true morphological nature of spidroin nanostructures in the precursor dope solution and the mechanisms by which these nanostructures transform into micrometer-scale silk fibers. Herein we determine the physical form of the natural spidroin precursor nanostructures stored within spider glands that seed the formation of their silks and reveal the fundamental structural transformations that occur during the initial stages of extrusion en route to fiber formation. Using a combination of solution phase diffusion NMR and cryogenic transmission electron microscopy (cryo-TEM), we reveal direct evidence that the concentrated spidroin proteins are stored in the silk glands of black widow spiders as complex, hierarchical nanoassemblies (∼300 nm diameter) that are composed of micellar subdomains, substructures that themselves are engaged in the initial nanoscale transformations that occur in response to shear. We find that the established micelle theory of silk fiber precursor storage is incomplete and that the first steps toward liquid crystalline organization during silk spinning involve the fibrillization of nanoscale hierarchical micelle subdomains.

Novel Highly Soluble Chimeric Recombinant Spidroins with High Yield.

Spider silk has been a hotspot in the study of biomaterials for more than two decades due to its outstanding mechanical properties. Given that spiders cannot be farmed, and their low silk productivity, many attempts have been made to produce recombinant spidroins as an alternative. Herein, we present novel chimeric recombinant spidroins composed of 1 to 4 repetitive units of aciniform spidroin (AcSp) flanked by the nonrepetitive N- and C-terminal domains of the minor ampullate spidroin (MiSp), all from Araneus ventricosus. The spidroins were expressed in the form of inclusion body in E. coli with high yield. Remarkably, the aqueous solubility of the four spidroins ranged from 13.4% to over 50% (m/v). The four spidroins could self-assemble into silk-like fibers by hand-drawing. The secondary structures of these proteins, determined by circular dichroism spectrum (CD) and Fourier transform infrared spectrum (FTIR), indicated a prominent transformation from α-helix to ß-sheet after fiber formation. The mechanical properties of the hand-drawn fibers showed a positive correlation with the spidroin molecular weight. In summary, this study describes promising biomaterials for further study and wide application.

Insights into the regulatory characteristics of silkworm fibroin gene promoters using a modified Gal4/UAS system.

The silkworm Bombyx mori is a valuable insect that synthesizes bulk amounts of fibroin protein in its posterior silk gland (PSG) and weaves these proteins into silk cocoons. The mechanism by which the fibroin protein is efficiently synthesized and precisely regulated is an important aspect that has yet to be fully elucidated. Here, we describe the regulatory characteristics of the promoters of fibroin protein-encoding genes, namely, fibroin heavy chain (fibH) and fibroin light chain (fibL), using an optimized Gal4/UAS binary system. We found that (1) UAS-linked enhanced green fluorescent protein (EGFP) was effectively activated in the PSGs of Gal4/UAS transgenic silkworms, and fluorescence was continuously detected in the PSGs after complete formation of silk glands. (2) In the PSGs of fourth- and fifth-instar larvae of transgenic silkworms driven by fibL-Gal4 (LG4) or fibH-Gal4 (HG4), EGFP mRNA was detected in only day-3 to day-6 fifth-instar larvae, while the EGFP protein could be detected at each day of both larval stages. (3) High-level expression of Gal4 and UAS-linked EGFP caused a delay in PSG degradation in Gal4/UAS transgenic silkworms. (4) At the early pupal stage, EGFP fluorescence was also detected in fat bodies of Gal4/UAS transgenic silkworms, indicating that the PSG-specific EGFP was transported into fat bodies during PSG degeneration; however, the underlying mechanism needs to be further elucidated. This study provides a modified Gal4/UAS system used for efficient tissue-specific expression of target genes in the PSGs of silkworms and provides new insights into the regulatory characteristics of the promoters of key fibroin protein-encoding genes.

CRISPR/Cas9 Initiated Transgenic Silkworms as a Natural Spinner of Spider Silk.

Using transgenic silkworms with their natural spinning apparatus has proven to be a promising way to spin spider silk-like fibers. The challenges are incorporating native-size spider silk proteins and achieving an inheritable transgenic silkworm strain. In this study, a CRISPR/Cas9 initiated fixed-point strategy was used to successfully incorporate spider silk protein genes into the Bombyx mori genome. Native-size spider silk genes (up to 10 kb) were inserted into an intron of the fibroin heavy or light chain (FibH or FibL) ensuring that any sequence changes induced by the CRISPR/Cas9 would not impact protein production. The resulting fibers are as strong as native spider silks (1.2 GPa tensile strength). The transgenic silkworms have been tracked for several generations with normal inheritance of the transgenes. This strategy demonstrates the feasibility of using silkworms as a natural spider silk spinner for industrial production of high-performance fibers.

Apoptosis of posterior silk gland of Bombyx mori during spinning period and the role of PI3K/Akt pathway.

Bombyx mori is an economic insect of the Lepidoptera. Its posterior silk gland (PSG) is an important organ for fibroin synthesis. In order to study the occurrence of apoptosis in PSG and the role of PI3K/Akt signaling pathway during spinning period, changes in morphology of silk gland, expressions of fibroin components Fib-H, Fib-L and P25 and Akt, TOR2, P70S6K and S6 in PI3K/Akt pathway, expressions of apoptosis related genes caspase-3, caspase-9 and activity of caspase-3 were explored. The results showed that the morphology of silk gland dramatically degenerated; transcription of Fib-H, Fib-L, and P25 gradually declined with time; and Fib-L protein level reduced by 0.6-fold at 72 h. Moreover, the transcription levels of Akt, TOR2, P70S6K, and S6 also decreased by 0.3-, 0.8-, 0.7-, and 0.1-fold, respectively, indicating that the downregulation of PI3K/Akt signaling pathway could lead to reduction in fibroin synthesis. In addition, the transcription levels of caspase-3 and caspase-9 increased by 1.3- and 3.6-fold, respectively, and the enzyme activity of caspase-3 grew at a maximum of 1.6-fold. The results showed the occurrence of apoptosis in PSG during spinning period. In conclusion, the present study indicated that both the decline in fibroin components and the increase in apoptosis-related genes were regulated by PI3K/Akt signaling pathway during spinning period, which shed new light on the functions of PI3K/Akt signaling pathway.

Recombinant Production, Characterization, and Fiber Spinning of an Engineered Short Major Ampullate Spidroin (MaSp1s).

Spider dragline silk exhibits an extraordinary toughness and is typically composed of two types of major ampullate spidroins (MaSp1 and MaSp2), differing in their proline content and hydrophobicity. In this paper, we recombinantly produced an unusual but naturally occurring short major ampullate spidroin (MaSp1s) as a fusion construct between established Latrodectus hesperus terminal domains and the novel Cyrtophora moluccensis core domain. The sequence of the recombinant spidroin was engineered to guarantee high yields upon recombinant production and was named eMaSp1s. Its solution structure as well as the mechanical properties of wet-spun eMaSp1s fibers were examined. Structural characterization using CD- and FTIR spectroscopy showed a predominantly α-helical solution structure and a high ß-sheet content within fibers. Surprisingly, eMaSp1s fibers show similar mechanical properties as wet-spun fibers of other engineered spider silk proteins, albeit eMaSp1s has a lower molecular weight and not the typical sequence repeats in its core domain. Therefore, the findings provide insights into the molecular interplay necessary to obtain the typical silk fiber mechanics.

A comparative investigation of Bombyx mori silk fibroin hydrogels generated by chemical and enzymatic cross-linking.

Fibroin, the major proteinaceous component of the silk fiber produced by larvae of the domesticated silk moth (Bombyx mori), has been widely investigated as a biomaterial for potential applications in tissue engineering and regenerative medicine. Following sol-gel transition, silk fibroin solutions can generate hydrogels that present certain advantages when employed as biomaterials, especially if they are cross-linked. The subject of this study was the self-cross-linking of silk fibroin through a process induced by the enzyme horseradish peroxidase (HRP) in the presence of hydrogen peroxide, a method only recently proposed and scarcely reported. The hydrogels were prepared either by physical cross-linking, by cross-linking with a natural compound (genipin), or by enzymatic cross-linking. The products were comparatively characterized in regard to their synthesis and background chemical aspects, physical and optical properties, mechanical properties, secondary structure, swelling/deswelling behavior, enzymatic degradation, and compatibility as substrates for cell adhesion and proliferation. The study confirmed the advantages of the HRP-induced cross-linking, which included considerably shorter gelation times, enhanced elasticity of the resulting hydrogels, and improved cytocompatibility. Discrepancies between certain results of this investigation and those reported previously were discussed in detail.

Dry-Spun Silk Produces Native-Like Fibroin Solutions.

Silk's outstanding mechanical properties and energy efficient solidification mechanisms provide inspiration for biomaterial self-assembly as well as offering a diverse platform of materials suitable for many biotechnology applications. Experiments now reveal that the mulberry silkworm Bombyx mori secretes its silk in a practically "unspun" state that retains much of the solvent water and exhibits a surprisingly low degree of molecular order (ß-sheet crystallinity) compared to the state found in a fully formed and matured fiber. These new observations challenge the general understanding of silk spinning and in particular the role of the spinning duct for structure development. Building on this discovery we report that silk spun in low humidity appears to arrest a molecular annealing process crucial for ß-sheet formation. This, in turn, has significant positive implications, enabling the production of a high fidelity reconstituted silk fibroin with properties akin to the gold standard of unspun native silk.

Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution.

The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate) as well as chimeric sequences (FlAS) in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi) that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution.

In planta production of ELPylated spidroin-based proteins results in non-cytotoxic biopolymers.

BACKGROUND: Spider silk is a tear-resistant and elastic biopolymer that has outstanding mechanical properties. Additionally, exiguous immunogenicity is anticipated for spider silks. Therefore, spider silk represents a potential ideal biomaterial for medical applications. All known spider silk proteins, so-called spidroins, reveal a composite nature of silk-specific units, allowing the recombinant production of individual and combined segments. RESULTS: In this report, a miniaturized spidroin gene, named VSO1 that contains repetitive motifs of MaSp1 has been synthesized and combined to form multimers of distinct lengths, which were heterologously expressed as elastin-like peptide (ELP) fusion proteins in tobacco. The elastic penetration moduli of layered proteins were analyzed for different spidroin-based biopolymers. Moreover, we present the first immunological analysis of synthetic spidroin-based biopolymers. Characterization of the binding behavior of the sera after immunization by competitive ELISA suggested that the humoral immune response is mainly directed against the fusion partner ELP. In addition, cytocompatibility studies with murine embryonic fibroblasts indicated that recombinant spidroin-based biopolymers, in solution or as coated proteins, are well tolerated. CONCLUSION: The results show that spidroin-based biopolymers can induce humoral immune responses that are dependent on the fusion partner and the overall protein structure. Furthermore, cytocompatibility assays gave no indication of spidroin-derived cytotoxicity, suggesting that recombinant produced biopolymers composed of spider silk-like repetitive elements are suitable for biomedical applications.

To spin or not to spin: spider silk fibers and more.

Spider silk fibers have a sophisticated hierarchical structure composed of proteins with highly repetitive sequences. Their extraordinary mechanical properties, defined by a unique combination of strength and extensibility, are superior to most man-made fibers. Therefore, spider silk has fascinated mankind for thousands of years. However, due to their aggressive territorial behavior, farming of spiders is not feasible on a large scale. For this reason, biotechnological approaches were recently developed for the production of recombinant spider silk proteins. These recombinant proteins can be assembled into a variety of morphologies with a great range of properties for technical and medical applications. Here, the different approaches of biotechnological production and the advances in material processing toward various applications will be reviewed.

Silk gland factor-2, involved in fibroin gene transcription, consists of LIM homeodomain, LIM-interacting, and single-stranded DNA-binding proteins.

SGF-2 binds to promoter elements governing posterior silk gland-specific expression of the fibroin gene in Bombyx mori. We purified SGF-2 and showed that SGF-2 contains at least four gene products: the silkworm orthologues of LIM homeodomain protein Awh, LIM domain-binding protein (Ldb), a sequence-specific single-stranded DNA-binding protein (Lcaf), and the silk protein P25/fibrohexamerin (fhx). Using co-expression of these factors in Sf9 cells, Awh, Ldb, and Lcaf proteins were co-purified as a ternary complex that bound to the enhancer sequence in vitro. Lcaf interacts with Ldb as well as Awh through the conserved regions to mediate transcriptional activation in yeast. Misexpression of Awh in transgenic silkworms induces ectopic expression of the fibroin gene in the middle silk glands, where Ldb and Lcaf are expressed. Taken together, this study demonstrates that SGF-2 is a multisubunit activator complex containing Awh. Moreover, our results suggest that the Ldb·Lcaf protein complex serves as a scaffold to facilitate communication between transcriptional control elements.

Transglutamination allows production and characterization of native-sized ELPylated spider silk proteins from transgenic plants.

In the last two decades it was shown that plants have a great potential for production of specific heterologous proteins. But high cost and inefficient downstream processing are a main technical bottleneck for the broader use of plant-based production technology especially for protein-based products, for technical use as fibres or biodegradable plastics and also for medical applications. High-performance fibres from recombinant spider silks are, therefore, a prominent example. Spiders developed rather different silk materials that are based on proteins. These spider silks show excellent properties in terms of elasticity and toughness. Natural spider silk proteins have a very high molecular weight, and it is precisely this property which is thought to give them their strength. Transgenic plants were generated to produce ELPylated recombinant spider silk derivatives. These fusion proteins were purified by Inverse Transition Cycling (ITC) and enzymatically multimerized with transglutaminase in vitro. Layers produced by casting monomers and multimers were characterized using atomic force microscopy (AFM) and AFM-based nanoindentation. The layered multimers formed by mixing lysine- and glutamine-tagged monomers were associated with the highest elastic penetration modulus.

Production of Bombyx mori silk fibroin incorporated with unnatural amino acids.

Silk fibroin incorporated with unnatural amino acids was produced by in vivo feeding of p-chloro-, p-bromo-, and p-azido-substituted analogues of L-phenylalanine (Phe) to transgenic silkworms (Bombyx mori) that expressed a mutant of phenylalanyl-tRNA synthetase with expanded substrate recognition capabilities in silk glands. Cutting down the content of Phe in the diet was effective for increasing the incorporation of Phe analogues but simultaneously caused a decrease of fibroin production. The azide groups incorporated in fibroin were active as chemical handles for click chemistry in both the solubilized and the solid (fibrous) states. The azides survived degumming in the boiling alkaline solution that is required for complete removal of the sericin layer, demonstrating that AzPhe-incorporated silk fibroin could be a versatile platform to produce "clickable" silk materials in various forms. This study indicates the huge potential of UAA mutagenesis as a novel methodology to alter the characteristics of B. mori silk.

An engineered Pichia pastoris platform for the biosynthesis of silk-based nanomaterials with therapeutic potential.

Silk fibroin (SF) from the cocoon of silkworm has exceptional mechanical properties and biocompatibility and is used as a biomaterial in a variety of fields. Sustainable, affordable, and scalable manufacturing of SF would enable its large-scale use. We report for the first time the high-level secretory production of recombinant SF peptides in engineered Pichia pastoris cell factories and the processing thereof to nanomaterials. Two SF peptides (BmSPR3 and BmSPR4) were synthesized and secreted by P. pastoris using signal peptides and appropriate spacing between hydrophilic sequences. By strain engineering to reduce protein degradation, increase glycyl-tRNA supply, and improve protein secretion, we created the optimized P. pastoris chassis PPGSP-8 to produce BmSPR3 and BmSPR4. The SF fed-batch fermentation titers of the resulting two P. pastoris cell factories were 11.39 and 9.48 g/L, respectively. Protein self-assembly was inhibited by adding Tween 80 to the medium. Recombinant SF peptides were processed to nanoparticles (NPs) and nanofibrils. The physicochemical properties of nanoparticles R3NPs and R4NPs from the recombinant SFs synthesized in P. pastoris cell factories were similar or superior to those of RSFNPs (Regenerated Silk Fibroin NanoParticles) originating from commercially available SF. Our work will facilitate the production by microbial fermentation of functional SF for use as a biomaterial.

Complex gene expression in the dragline silk producing glands of the Western black widow (Latrodectus hesperus).

BACKGROUND: Orb-web and cob-web weaving spiders spin dragline silk fibers that are among the strongest materials known. Draglines are primarily composed of MaSp1 and MaSp2, two spidroins (spider fibrous proteins) expressed in the major ampullate (MA) silk glands. Prior genetic studies of dragline silk have focused mostly on determining the sequence of these spidroins, leaving other genetic aspects of silk synthesis largely uncharacterized. RESULTS: Here, we used deep sequencing to profile gene expression patterns in the Western black widow, Latrodectus hesperus. We sequenced millions of 3'-anchored "tags" of cDNAs derived either from MA glands or control tissue (cephalothorax) mRNAs, then associated the tags with genes by compiling a reference database from our newly constructed normalized L. hesperus cDNA library and published L. hesperus sequences. We were able to determine transcript abundance and alternative polyadenylation of each of three loci encoding MaSp1. The ratio of MaSp1:MaSp2 transcripts varied between individuals, but on average was similar to the estimated ratio of MaSp1:MaSp2 in dragline fibers. We also identified transcription of TuSp1 in MA glands, another spidroin family member that encodes the primary component of egg-sac silk, synthesized in tubuliform glands. In addition to the spidroin paralogs, we identified 30 genes that are more abundantly represented in MA glands than cephalothoraxes and represent new candidates for involvement in spider silk synthesis. CONCLUSIONS: Modulating expression rates of MaSp1 variants as well as MaSp2 and TuSp1 could lead to differences in mechanical properties of dragline fibers. Many of the newly identified candidate genes likely encode secreted proteins, suggesting they could be incorporated into dragline fibers or assist in protein processing and fiber assembly. Our results demonstrate previously unrecognized transcript complexity in spider silk glands.

Control of amyloid assembly by autoregulation.

The assembly of proteins into amyloid fibrils can be an element of both protein aggregation diseases and a functional unit in healthy biological pathways. In both cases, it must be kept under tight control to prevent undesired aggregation. In normophysiology, proteins can self-chaperone amyloidogenic segments by restricting their conformational flexibility in an overall stabilizing protein fold. However, some aggregation-prone segments cannot be controlled in this manner and require additional regulatory elements to limit fibrillation. The present review summarizes different molecular mechanisms that proteins use to control their own assembly into fibrils, such as the inclusion of a chaperoning domain or a blocking segment in the proform, the controlled release of an amyloidogenic region from the folded protein, or the adjustment of fibrillation propensity according to pH. Autoregulatory elements can control disease-related as well as functional fibrillar protein assemblies and distinguish a group of self-regulating amyloids across a wide range of biological functions and organisms.

Silk layering as studied with neutron reflectivity.

Neutron reflectivity (NR) measurements of ultrathin surface films (below 30 nm) composed of Bombyx mori silk fibroin protein in combination with atomic force microscopy and ellipsometry were used to reveal the internal structural organization in both dry and swollen states. Reconstituted aqueous silk solution deposited on a silicon substrate using the spin-assisted layer-by-layer (SA-LbL) technique resulted in a monolayer silk film composed of random nanofibrils with constant scattering length density (SLD). However, a vertically segregated ordering with two different regions has been observed in dry, thicker, seven-layer SA-LbL silk films. The vertical segregation of silk multilayer films indicates the presence of a different secondary structure of silk in direct contact with the silicon oxide surface (first 6 nm). The layered structure can be attributed to interfacial ß-sheet crystallization and the formation of well-developed nanofibrillar nanoporous morphology for the initially deposited silk surface layers with the preservation of less dense, random coil secondary structure for the layers that follow. This segregated structure of solid silk films defines their complex nonuniform behavior in the D(2)O environment with thicker silk films undergoing delamination during swelling. For a silk monolayer with an initial thickness of 6 nm, we observed the increase in the effective thickness by 60% combined with surprising decrease in density. Considering the nanoporous morphology of the hydrophobic silk layer, we suggested that the apparent increase in its thickness in liquid environment is caused by the air nanobubble trapping phenomenon at the liquid-solid interface.

Silk Fibroin as a Bioink - A Thematic Review of Functionalization Strategies for Bioprinting Applications.

Bioprinting is an emerging tissue engineering technique that has attracted the attention of researchers around the world, for its ability to create tissue constructs that recapitulate physiological function. While the technique has been receiving hype, there are still limitations to the use of bioprinting in practical applications, much of which is due to inappropriate bioink design that is unable to recapitulate complex tissue architecture. Silk fibroin (SF) is an exciting and promising bioink candidate that has been increasingly popular in bioprinting applications because of its processability, biodegradability, and biocompatibility properties. However, due to its lack of optimum gelation properties, functionalization strategies need to be employed so that SF can be effectively used in bioprinting applications. These functionalization strategies are processing methods which allow SF to be compatible with specific bioprinting techniques. Previous literature reviews of SF as a bioink mainly focus on discussing different methods to functionalize SF as a bioink, while a comprehensive review on categorizing SF functional methods according to their potential applications is missing. This paper seeks to discuss and compartmentalize the different strategies used to functionalize SF for bioprinting and categorize the strategies for each bioprinting method (namely, inkjet, extrusion, and light-based bioprinting). By compartmentalizing the various strategies for each printing method, the paper illustrates how each strategy is better suited for a target tissue application. The paper will also discuss applications of SF bioinks in regenerating various tissue types and the challenges and future trends that SF can take in its role as a bioink material.