Abstract
Recombinant human collagen (rhCol) holds broad potential in biomedical and industrial applications due to its high purity and low immunogenicity. However, large-scale production of structurally stable and functionally active rhCol remains challenging. A novel strategy integrating collagen sequence optimization and microbial prolyl-4-hydroxylase (P4H) screening was developed to enable efficient production of triple-helical rhCol in Komagataella phaffii. Five Type III collagen variants (ColP1 ~ ColP5) were rationally designed based on interchain salt-bridge engineering to improve structural stability and biological activity, with ColP2 showing superior expression and functionality. A systematic evaluation of four microbial P4Hs identified Bacillus megaterium P4H (BmP4H) as the most effective catalyst for proline hydroxylation, enabling stable triple-helix formation. Combined with strain optimization, promoter and signal peptide screening, and 5-L scale fermentation, this approach achieved a high rhCol yield of 2.54 g/L with confirmed triple-helical structure. These results demonstrate an integrated and scalable platform for high-level production of functional recombinant collagen, providing a promising foundation for its industrial and clinical applications. Key Points • Co-expression of BmP4H enables stable triple-helical collagen in yeast. • Strain X-33, promoter P(AOX1), and a-factor leader optimize collagen secretion. • Scale-up in 5L bioreactor achieves 2.54 g/L rhCol production.