3,4-Dihydroxyphenylalanine (DOPA),
a naturally occurring yet noncanonical
amino acid, endows protein polymers with diverse chemical reactivities
and novel functionalities. Although many efforts have been made to
incorporate DOPA into proteins, the incorporation efficiency and production
titer remain low and severely hinder the exploration of these peculiar
proteins for biomaterial fabrication. Here, we report an efficient
biosynthetic strategy to produce large amounts of DOPA-incorporated
structural proteins for the fabrication of hydrogels with tunable
mechanical properties. First, synthetic genes were constructed that
encode repetitive resilin-like proteins (RLPs) with varying proportions
of tyrosine residues and molecular weights (Mw). Decoding of these genes into RLPs incorporated with DOPA
was achieved via mis-aminoacylation of DOPA by endogenous tyrosyl-tRNA
synthetase (TyrRS) in recombinant Escherichia coli cells. By developing a stoichiometry-guided two-phase culture strategy,
we achieved independent control of the bacterial growth and protein
synthesis phases. This enabled hyperproduction of the DOPA-incorporated
RLPs at gram-per-liter levels and with a high DOPA incorporation yield
of 76–85%. The purified DOPA-containing RLPs were then successfully
cross-linked into bulk hydrogels via facile DOPA–Fe3+ complexations. Interestingly, these hydrogels exhibited viscoelastic
and self-healing properties that are highly dependent on the catechol
content and Mw of the RLPs. Finally, exploration
of the molecular cross-linking mechanisms revealed that higher DOPA
contents of the proteins would result in the concomitant occurrence
of metal coordination and oxidative covalent cross-linking. In summary,
our results suggest a useful platform to generate DOPA-functionalized
protein materials and provide deeper insights into the gelation systems
based on DOPA chemistry.