Structural and mechanical properties of engineered silkworm-spider composite silk.

Spider silk demonstrates significant potential for biomaterials and medicinal applications owing to its favorable mechanical properties and biocompatibility. However, spiders are difficult to raise on a large scale, and obtaining silk proteins directly from spiders is inefficient and expensive. A promising strategy for addressing these challenges involves expressing spider silk proteins in transgenic silkworms. In this study, transcription activator-like effector nuclease (TALEN)-mediated genome-targeted editing was employed to separately fuse 1-, 2-, 4-, and eightfold repeats of the cre-MaSp1 gene from black widow spiders to the sericin 1 gene. AlphaFold 3 structure prediction and infrared spectroscopy showed that the β-sheet and helix contents of the composite silk proteins progressively increased with the increase in the number of fused cre-MaSp1 repeats. Mechanical property testing showed that the maximum stress and maximum strain of the silkworm-spider composite silk containing the eightfold cre-MaSp1/Ser1 fusion protein were 39.4 % and 62.2 % higher than those of the wild-type, respectively, representing the best performance among all the lines. This study provides insights into sericin modification and further confirms that the expression of the cre-MaSp1 gene harboring a large number of repeats can improve the mechanical properties of silkworm silk. STATEMENT OF SIGNIFICANCE: Silkworm silk is a kind of natural protein fiber, and the improvement of silk performance is a long-term focus. This study aims to improve the mechanical properties of silk by endowing it with functional proteins through targeted modification of silk proteins. Four different repeats of cre-MaSp1 gene from black widow spiders separately fused into endogenous Ser1 by TALEN-mediated homology-directed recombination. The fusion proteins were successfully expressed and secreted into the cocoon shell. Tensile testing indicated that eightfold cre-MaSp1 repeats significantly increased the maximum stress and strain of the composite silk by 39.4 % and 62.2 % over the wild-type, respectively. Our work provides insights into improving silk properties and expands the potential applications of the silkworm silk gland bioreactor.