Recombinant production of silk fibroin in Nicotiana benthamiana using a modular library for transgene assembly.
Year: 2025
Type: article
Abstract: 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 high-quality, 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. • Developed a versatile modular vector library for efficient assembly of large repetitive DNA sequences. • Produced high-molecular-weight fibroin polymers up to 172 kDa in N. benthamiana . • Recombinant fibroin coatings showed suitable water contact angle and adhesion strength for medical use. • Produced resilin polymers of over 250 kDa confirming the versatility of the library. • Created chimeric fibroin-resilin polymers for tunable biomaterials.
Source: New Biotechnology
Institution: Maastricht University
Language: English
Subfield: Biotechnology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,670
OpenAlex ID: W4415896901