Work

Enhanced biosynthesis of full-length triple-helical human type III collagen in engineered Komagataella phaffii

Year: 2026

Type: article

Abstract: Fibrillar type III collagen, characterized by a distinct triple helix structure, is a critical structural component of the mammalian extracellular matrix (ECM). Its unique mechanical properties and biological functions make it a highly valuable biomaterial for medical applications. However, the limited availability of natural human type III collagen (hCOL3) and the defects of animal-derived sources necessitate sustainable alternatives. Here, we developed an integrated molecular engineering strategy for the efficient production of full-length recombinant human type III collagen (rhCOL3) in Komagataella phaffii. The N- and C-terminal propeptides of the rhCOL3α1 chain were rationally redesigned to preserve its intrinsic capability for triple helix formation. Synergistic co-expression of human P4Hαβ and Bacillus anthracis-derived BaP4H achieved a proline hydroxylation level of 52.88%, enabling the stable assembly of rhCOL3 procollagen homotrimers (> 300 kDa). Subsequent optimization, including high-copy-number strain screening, signal sequence substitution (with Ost1 identified as optimal), and co-expression of the molecular chaperones (e.g., BCY1 and ERO1), significantly enhanced productivity. The engineered strain achieved a rhCOL3 procollagen yield of 430 mg/L in a 5-L fed-batch bioreactor. The purified rhCOL3 exhibited a stable triple helix conformation, a melting temperature of 38.87 °C, and the ability to self-assemble into fibrils with a characteristic D-banding pattern. Biological assays confirmed that the rhCOL3 promotes cell proliferation, adhesion, and migration at levels comparable to those of natural collagen. This study establishes a promising platform for the industrial-scale production of bioactive, triple-helical human collagen. KEY POINTS: • Synergistic P4Hαβ and BaP4H co-expression achieves 52.88% Hyp level and Tm 38.87 °C. • Ost1 and Bcy1 co-expression elevated rhCOL3 yield to 430 mg/L in a 5-L bioreactor. • Purified rhCOL3 self-assembles into D-banded fibrils resembling native collagen.

Source: Applied Microbiology and Biotechnology

DOI: 10.1007/s00253-026-13996-7