A Novel Transgenic Silkworm Line for Mass Production of Azido-Incorporated Silk Fiber

Abstract

Bombyx mori silk is a sustainable bioresource with diverse applications in textiles, electronics, and medicine. It attracts much research interest because of its mechanical toughness, controllable biodegradability, electrical properties, and broad biocompatibility. A novel silk type, AzidoSilk, was developed based on genetic code expansion (GCE) methodology: an emerging biotechnology that can expand the repertoire of amino acid building blocks used for protein biosynthesis. An azido-bearing synthetic amino acid, 4-azidophenylalanine (AzPhe), is incorporated in AzidoSilk. Because the azido group in AzPhe can be modified selectively with functional molecules by click chemistry reactions, AzidoSilk can be a versatile platform for producing functional silk-based materials for various applications. Unfortunately, low production of AzidoSilk by a current transgenic B. mori line hampers its practical applications. This report describes breeding of a novel transgenic B. mori line for mass production of AzidoSilk using a conventional backcrossing method from the current transgenic line H06 to the high silk-producing wildtype strain MCS4. The resultant line H06-MCS4 established after five times of backcrossing produced 2.6 times greater amounts of AzidoSilk per larva with 25% increased productivity. Although AzPhe incorporation in silk fibroin decreased by 21%, clear and selective modification of azido groups in AzidoSilk were verified using click chemistry reaction tests. The AzidoSilk fiber was prepared using standard machine reeling. Its mechanical properties were found to be equivalent to those of normal silk fiber from MCS4. The generation of H06-MCS4 will open possibilities for producing AzidoSilk on an industrial scale.