Optimizated Prolyl‐4‐Hydroxylase Expression in Komagataella phaffii Enables Efficient Triple‐Helix Formation of Recombinant Human Collagen

ABSTRACT Prolyl‐4‐hydroxylase (P4H) catalyzes the hydroxylation of proline to hydroxyproline, a critical post‐translational modification essential for the formation and stability of collagen's triple‐helix structure. Although indispensable for recombinant human collagen (rhCol) production, P4H suffers from low expression levels, which significantly limit its industrial application. In this study, we achieved high‐level expression of P4H in Komagataella phaffii ( K. phaffii ), enabling the formation of the triple‐helix structure of rhCol. Initially, six codon‐optimized P4H genes were expressed in K. phaffii GS115, among which the Bacillus anthracis ‐derived BaP4H showed the highest expression level and was correctly localized to the endoplasmic reticulum, where efficient hydroxylation of collagen was facilitated. Subsequently, host strains screening identified K. phaffii X33 as the most productive, with an enzymatic activity of 6.64 × 10 3 U/L, representing to an 15.3% improvement over GS115. Further enhancement through promoter engineering and genomic integration site optimization increased enzymatic activity by 20.9%, reaching 8.03 × 10 3 U/L. Additional improvements via co‐expression of molecular chaperones and fusion with solubility‐enhancing tags elevated activity to 1.35 × 10⁴ U/L. Finally, co‐expression of the BaP4H with rhCol enabled the successful formation of the triple‐helical structure, which was not observed with rhCol expressed alone. This study establishes an effective strategy for optimizing P4H expression and enables the functional biosynthesis of recombinant collagen, with promising potential for biomedical and industrial applications.