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Abstract <p>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 <i>Komagataella phaffii</i>. 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 <i>Bacillus megaterium</i> 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&#xa0;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. <Table Float="No" ID="Taba"> <tgroup cols="2"> <colspec align="left" colname="c1" colnum="1" /> <colspec align="left" colname="c2" colnum="2" /> <tbody> <row> <entry nameend="c2" namest="c1"> <p><b>Key Points</b></p> <p>• <i>Co-expression of BmP4H enables stable triple-helical collagen in yeast.</i></p> <p>• <i>Strain X-33, promoter P</i><sub><i>AOX1</i></sub><i>, and a-factor leader optimize collagen secretion.</i></p> <p>• <i>Scale-up in 5L bioreactor achieves 2.54&#xa0;g/L rhCol production.</i></p> </entry> </row> </tbody> </tgroup> </Table></p>

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Efficient secretory expression of type III recombinant human collagen with triple-helical structure in Komagataella phaffii

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年9月3日

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  • Yaqian Ma,
  • Yang Li,
  • Nan Wang,
  • Chenxiao Han,
  • Qisheng Liu,
  • Liqin Sun,
  • Zhuqing Ma,
  • Hailing Zhang

摘要

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 PAOX1, and a-factor leader optimize collagen secretion.

• Scale-up in 5L bioreactor achieves 2.54 g/L rhCol production.