Abstract

TyrA knockout enables DOPA incorporation via TyrRS. • FP1 achieves residue-specific DOPA incorporation and 18.01 mg L⁻¹ purified yield. • FP3 reaches 90% solubility via TrxA fusion. • MagR fusion further improves FP3 solubility to 85%. • Two-stage cultivation enhances recombinant protein yield. . Mussel foot proteins (Mfps) achieve exceptional marine adhesion through post-translational conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA). Recombinant production, however, is limited by poor solubility, low yields, and insufficient DOPA incorporation. We generated a genetically stable tyrA -deficient Escherichia coli chassis using CRISPR/Cas, thereby abolishing endogenous tyrosine biosynthesis and enabling residue-specific in vivo incorporation of exogenously supplied DOPA into mussel foot protein (MFP) mimics through selective pressure incorporation (SPI). A two-stage cultivation strategy decoupled biomass accumulation from DOPA-dependent protein synthesis, yielding 18.01 mg L⁻¹ of purified FP1. Residue-specific DOPA incorporation was verified by the characteristic + 16 Da shift in the [M−H]⁻ ion, accompanied by the loss of the tyrosine signal following exogenous DOPA supplementation in M9 medium. FP3 was largely soluble (65%) in crude extracts, while FP5 expression remained minimal. Fusion to thioredoxin (TrxA) and magnetoreceptor protein (MagR) further enhanced FP3 solubility to 90% and 85%, respectively. Proteins expressed in minimal M9 medium displayed exceptional shear stability, with viscosity fluctuations limited to ±2.2%, reflecting preserved catechol chemistry and structural integrity. This integrated strategy overcomes recurring trade-offs between DOPA incorporation, solubility, and yield, providing a basis for the potentially scalable production of functional, catechol-rich Mfps. Collectively, these findings support the development of next-generation mussel-inspired adhesives and catechol-based biomaterials.