Engineering Prolyl 4-Hydroxylation for High-Performance Recombinant Collagen Production: From Molecular Design and Microbial Cell Factories to Industrial Translation

Recombinant collagen is an attractive alternative to animal-derived collagen for applications in tissue engineering, wound repair, regenerative medicine, and advanced aesthetic biomaterials. However, many microbial production hosts lack sufficient endogenous prolyl 4-hydroxylase (P4H) activity, resulting in inadequate proline hydroxylation, reduced triple-helix stability, and potentially impaired material performance. Engineering P4H therefore offers a promising strategy for enhancing proline hydroxylation and improving the triple-helix stability of recombinant collagen. This review summarizes recent advances in the structural characterization, catalytic mechanisms, and engineering of P4H systems from animal, plant, viral, and microbial sources. Particular attention is given to the structural and functional differences between animal-derived α2β2 tetrameric P4Hs and structurally simpler monomeric P4Hs, including their distinct requirements for subunit assembly, folding, substrate recognition, and heterologous expression. Structure-guided engineering strategies, including active-site remodeling, substrate-binding-pocket expansion, flexible-loop engineering, collagen-anchoring module fusion, and computationally guided protein design, are evaluated for their potential to improve hydroxylation efficiency toward long-chain collagen substrates and reduce nonproductive uncoupling. At the host–cell level, synthetic biology strategies for coordinating P4H expression, subunit assembly, chaperone-assisted folding, secretion, and cofactor metabolism are discussed. The review also highlights the importance of engineering α-ketoglutarate (α-KG), iron, and L-ascorbate metabolism to match cofactor availability with catalytic demand while minimizing cellular stress. In addition, the spatial and temporal coordination of collagen synthesis, P4H-mediated hydroxylation, intracellular folding, and secretion is discussed as a means of improving hydroxylation efficiency and product uniformity. A comprehensive quality-assessment framework is further proposed, integrating site-specific mass spectrometry for hydroxylation profiling, spectroscopic and thermal analyses for structural characterization, and cell-based assays for evaluating biological activity. Finally, the review addresses challenges associated with industrial scale-up, including oxygen-transfer limitations, dynamic cofactor management, process consistency, and regulatory compliance. Emerging approaches involving artificial intelligence, cell-free protein synthesis, and integrated computational–experimental workflows are also considered for accelerating the development of clinically and industrially viable recombinant collagen products.