Condensate-based sequestration enables high-efficiency synthesis of mussel foot protein Mcofp-3

ABSTRACT Mussel foot proteins (Mfps) hold immense potential for medicine and bioengineering, owing to their exceptional adhesion and biocompatibility. However, traditional extraction from mussels is low-yield and ecologically unsustainable. While microbial synthesis offers a green alternative, the intrinsic hydrophobicity and adhesiveness of Mfps often induce severe host cytotoxicity, resulting in low yields. Initially, attempts to express Mcofp-3 in Escherichia coli were limited to 60 mg L −1 due to such cytotoxic effects. To overcome this bottleneck, we developed a hyaluronic acid (HA)-driven “condensate-based sequestration” strategy. This approach leverages electrostatic interactions to sequester Mcofp-3 into condensates, thereby shielding the host cells and alleviating metabolic stress. This strategy significantly restored cell viability, increasing the titer to 460 mg L −1 in shake flasks. Moreover, the process was successfully scaled up to a 50 L fed-batch fermentation, achieving a final titer of 1.6 g L −1 . Following production, in vitro 3,4-dihydroxyphenylalanine (DOPA) modification was performed using immobilized TyrVs-CipA, yielding functional Mcofp-3 that exhibited anti-inflammatory activity and enhanced cell migration capabilities. This work establishes a versatile, green paradigm for the high-yield production of proteins with low yields and increased toxicity in bacterial expression systems. IMPORTANCE Mussel foot proteins (Mfps) are prized bio-adhesives for medical applications, yet their supply remains limited. Harvesting mussels is environmentally unsustainable, while microbial synthesis is challenging due to the severe toxicity of these proteins to host cells. In this study, we developed a “condensate-based sequestration” strategy to overcome this bottleneck. By employing hyaluronic acid to isolate the toxic proteins within the bacteria, we shielded the host cells and achieved high-yield production. This approach enabled the gram-scale synthesis of modified Mfps with desirable bioactivity. Beyond synthesizing Mfps, this strategy establishes a versatile, green platform for manufacturing a wide range of other “difficult-to-express” toxic proteins that are currently commercially unviable.