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

IF 4.3 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Applied Microbiology and Biotechnology Pub Date : 2025-09-03 DOI:10.1007/s00253-025-13566-3

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

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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.

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三螺旋结构重组人ⅲ型胶原蛋白的高效分泌表达

重组人胶原蛋白（rhCol）因其高纯度和低免疫原性而在生物医学和工业应用中具有广泛的潜力。然而，大规模生产结构稳定和功能活跃的rhCol仍然具有挑战性。研究了一种结合胶原蛋白序列优化和微生物P4H筛选的新策略，以实现法菲Komagataella phaffii三螺旋rhCol的高效生产。基于链间盐桥工程合理设计了5种III型胶原变体ColP1 ~ ColP5，以提高其结构稳定性和生物活性，其中ColP2表现出更优越的表达和功能。通过对四种微生物P4H的系统评价，发现巨型芽孢杆菌（Bacillus megaterium P4H, BmP4H）是脯氨酸羟基化最有效的催化剂，可以稳定地形成三螺旋结构。结合菌株优化、启动子和信号肽筛选、5-L规模发酵，该方法获得了高rhCol产量2.54 g/L，并确定了三螺旋结构。这些结果为高水平生产功能性重组胶原提供了一个集成的、可扩展的平台，为其工业和临床应用提供了良好的基础。

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来源期刊

Applied Microbiology and Biotechnology 工程技术-生物工程与应用微生物

CiteScore

10.00

自引率

4.00%

发文量

535

审稿时长

2 months

期刊介绍： Applied Microbiology and Biotechnology focusses on prokaryotic or eukaryotic cells, relevant enzymes and proteins; applied genetics and molecular biotechnology; genomics and proteomics; applied microbial and cell physiology; environmental biotechnology; process and products and more. The journal welcomes full-length papers and mini-reviews of new and emerging products, processes and technologies.