A hydroxylated collagen-like construct with an integrin-binding motif produced in a probiotic chassis: Synthesis, structural stability, and in-vitro bioactivity.

Recombinant production of collagen in Escherichia coli is pivotal for advancing biomedical applications, yet it is frequently hampered by critical challenges, notably endotoxin contamination and insufficient prolyl hydroxylation. To address these limitations, we engineered the probiotic bacterium E. coli Nissle 1917 (EcN) as a chassis for producing a hydroxylated human type III collagen-like protein named R8. Through the co-expression of R8 with Bacillus anthracis prolyl 4-hydroxylase (BaP4H) in EcN, we achieved a yield of 0.26 mg/mL for the hydroxylated collagen. A hydroxylation rate of 60 % was achieved, with LC-MS/MS mapping confirming modification at 33 out of 65 proline residues. Hydroxylated R8 exhibits enhanced thermal stability, maintaining the structural integrity of its triple helix and assembling into a porous fibrous network. Crucially, R8 from EcN showed reduced immunogenicity in macrophage activation assays, in stark contrast to material from conventional E. coli BL21(DE3). Moreover, hydroxylated R8 exhibits excellent biocompatibility, significantly promoting fibroblast proliferation and migration, and underscoring the critical role of this modification. This study establishes a strategy for producing bioactive collagen, whilst highlighting the critical importance of hydroxylation for collagen stability and function.