Recombinant production of silk fibroin in Nicotiana benthamiana using a modular library for transgene assembly

Fibroin, the primary component of silkworm silk, is a natural biocompatible and biodegradable material known for its exceptional mechanical properties, making it highly valued in the biomedical field. Traditional methods of extracting fibroin from Bombyx mori silk present challenges related to quality and purity of the product, as well as sustainability and ethics of the process. This underscores the need for a recombinant platform for producing highquality, monodisperse vegan fibroin. The production of fibroin is particularly challenging due to its large and repetitive nature, also at the nucleic acid level. To address this challenge, we developed a DNA-mediated assembly method that utilizes a vector library offering flexibility to test the expression of recombinant proteins in different subcellular localizations in plants and the combination of various protein modules. This approach involves a series of Golden Gate cloning steps to gradually assemble the fibroin coding sequence, starting from the smallest repetitive unit. Using this method, we successfully produced recombinant fibroin polymers of up to 172 kDa in Nicotiana benthamiana via agroinfiltration. Additionally, we demonstrated the versatility of our vector library by assembling genetic constructs for another repetitive protein, resilin, and by creating chimeric proteins.