Engineering silkworm-spider composite silk via the fibroin light chain fusion system in Bombyx mori.

Spider silk possesses excellent material properties and demonstrates significant potential for application in the fields of biomedicine and the military. However, the cannibalistic and territorial nature of spiders precludes farm-based mass production of spider silk. To overcome this limitation, we employed targeted genome editing to enable the biosynthetic production of spider silk proteins in silkworm silk gland bioreactors. Specifically, one fold and double repeats of the cre-MaSp1 gene from the black widow spider were inserted into the 3' end of the silkworm FibL gene via site-directed integration using transcription activator-like effector nuclease (TALEN)-mediated homology-directed repair. By introducing the T2A self-cleavage peptide, which allows normal cross-linking of intact FibL to FibH, cre-MaSp1 are efficiently secreted into the cocoon shell, while mitigating the damage to cocoon economic traits caused by previous FibL direct fusion. The tensile test results showed that the expression of the cre-MaSp1 gene was beneficial for changing the maximum strain of the resulting transgenic silk fibers. Notably, as the number of repeats increases, the maximum strain and stress of the transgenic silk shows a rising trend, pointing the way toward improved mechanical properties of the silk. Furthermore, AlphaFold 3 prediction and Fourier-transform infrared (FTIR) spectroscopic analyses indicated that the transgenic silk contained a higher content of α-helix and random coil structures than wild-type silk. RNA sequencing results further suggested that the integration of the cre-MaSp1 gene into the FibL locus of the silkworm interfered with ribosome biogenesis. This study provides a promising strategy for the biofabrication of silkworm-spider composite silk.