An efficient microbial secretion platform for developing supercharged polypeptides-based functional materials from C2 feedstock

Nature-inspired protein materials boast superior mechanical and biological properties, but efficient and sustainable production remains a challenge. Here, we present a microbial secretion platform in Corynebacterium glutamicum for the production of supercharged polypeptides (SUPs) using C2 feedstock as the primary carbon source, featuring a simplified purification process for the development of functional protein materials. SUPs with the Val-Pro-Gly-Lys-Gly (VPGKG)ₙ motif were successfully transported extracellularly via the Sec-dependent pathway; however, increasing the number of repeat units resulted in expression instability and truncation. Optimized codon designs with reduced unit sequence homology, significantly stabilizing expression and enabling efficient secretion of SUP K72 (VPGVG(VPGKG)₉)₈. Through comparative analyses of host strains, promoters, and carbon sources, we identified the ATCC13032/A256/Acetate (3 A system) as the optimal platform for large-scale K72 production. This system, specifically activated during the glyoxylate cycle under acetate metabolism, achieved K72 secretion levels that were twice of the traditional P tac /IPTG system. Following systematic optimization of bioprocess parameters, including medium composition and fermentation conditions, production was successfully scaled up to a 5-L bioreactor, achieving a final titer of 3.67 g/L—far surpassing the previously reported 0.2–0.5 g/L in Escherichia coli using a 100-L fermenter. After chromatography-free purification, K72 was fabricated into high-strength protein fibers with an ultimate tensile strength of 308.97 ± 43.76 MPa. Additionally, an ultra-strong bio-glue was developed, achieving a fracture strength of 14.08 ± 1.26 MPa on hard substrates. This study demonstrates a sustainable approach for producing proteins from C2 feedstock, providing a cost-effective solution to advance the commercialization of protein-based materials.