Design and Heterologous Production of Bagworm Silk-Inspired Proteins in Aspergillus oryzae.

Silk-inspired proteins are attractive biomacromolecules owing to their structural diversity, sustainability, and tunable physicochemical properties. Among natural silks, the bagworm silk produced by Eumeta variegata has attracted increasing attention because of its unique sequence architecture and remarkable reported mechanical performance. However, the recombinant production of bagworm silk-inspired proteins remains largely unexplored. In this study, we designed and heterologously expressed 2 bagworm silk-inspired protein variants in Aspergillus oryzae using a synthetic expression system. The variants comprised a representative bagworm silk with an alanine- and glycine-rich repeat motif flanked by carbohydrate-binding modules at the N- and C-termini. Expression screening in engineered A. oryzae strains demonstrated stable accumulation of C-tag-reactive species corresponding to both variants in a protease-deficient host, whereas comparable accumulation was not detected in a carbon-catabolite-repressor-deficient strain under the tested conditions. The selected variant was further produced in a 2-l bioreactor, demonstrating the feasibility of scale-up and establishing A. oryzae as a promising platform for bagworm silk-inspired protein production. The recovered H-eBGW-containing protein fractions were readily processed into thin films and exhibited temperature-dependent changes in mechanical properties and surface wettability, demonstrating the processability and curing-responsive behavior of the recovered material. Collectively, our results demonstrate the feasibility of producing structurally complex bagworm silk-inspired proteins in filamentous fungi and provide insights into the design, expression, and processing of recombinant silk-inspired proteins for future bio-based applications.