Mass spider silk production through targeted gene replacement in <i>Bombyx mori</i>.

Abstract

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, <i>Bombyx mori</i>, 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 <i>B. mori</i> by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (<i>FibH</i>) with the major ampullate spidroin-1 gene (<i>MaSp1</i>) in the spider <i>Nephila clavipes</i> We successfully replaced the ∼16-kb endogenous <i>FibH</i> gene with a 1.6-kb <i>MaSp1</i> gene fused with a 1.1-kb partial <i>FibH</i> 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 <i>MaSp1</i> 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.