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Efficient heterologous expression of bovine lactoferrin in Pichia pastoris and characterization of antibacterial activity

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Abstract

The low-expression level of lactoferrin (LF) in the production process poses a significant challenge. This study aimed to efficiently express bovine lactoferrin (BLF) using Pichia pastoris GS115 as the expression host and PIC9K as the recombinant vector. Optimization strategies included codon usage, promoter selection, and fermentation conditions. The blf gene was optimized for P. pastoris GS115 bias, resulting in the construction of the recombinant vector pPIC9K-UBLF1-3 controlled by the AOX1 promoter. SDS-PAGE analysis revealed soluble and efficient expression of ublf3 in P. pastoris GS115, with a molecular mass of approximately 76 kDa. The transformant P. pastoris GS115/pGAP9K-UBLF3-4 resistant at 4 mg·mL−1 G418, exhibited a ublf3 gene copy number of 5.88 through high-copy screening. Optimal expression conditions of recombinant UBLF were determined as 24℃, pH 5.0 and 220 r·min−1 through fermentation condition optimization. Under these conditions, recombinant UBLF production reached 40.62 mg·L−1. The yield of recombinant UBLF was reached 824.93 mg·L−1 through high-density fermentation. Antibacterial assay demonstrated the efficacy of recombinant UBLF against Escherichia coli JM109 and Staphylococcus aureus CGMCC 1.282. This study successfully achieved the efficient heterologous expression of recombinant UBLF in P. pastoris GS115, providing valuable insight for industrial production and the potential development of natural antibacterial agents.

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Acknowledgements

We are grateful to the Lab of Brewing Microbiology and Applied Enzymology, School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University in encouraging us for the studies.

Funding

This project was supported by the National Key Research and Development Program of China (2023YFA0914500), the National Science Foundation of China (32271487), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-12), and the Program of Introducing Talents of Discipline to Universities (111-2-06).

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  1. Rongzhen Zhang

    Present address: School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi City, 214122, China

Authors and Affiliations

  1. Lab of Brewing Microbiology and Applied Enzymology, School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi City, 214122, People’s Republic of China

    Xinyi Zhang, Zhiwen Xi, Huiting Zhao, Yan Xu & Rongzhen Zhang

  2. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Wenchi Zhang

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  1. Xinyi Zhang
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Contributions

XY conducted experiments, analyzed and interpreted data, and wrote the draft. ZW and HT helped with the data analysis and performed the calculation. RZ led the project and revised the manuscript. YX and WC revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Rongzhen Zhang.

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Yan Xu as the Editor-in-Chief of SMAB, the authors declare a conflict of interest for this paper. Yan Xu has been excluded from the peer review and decision-making process to ensure unbiased evaluation and strict adherence to ethical guidelines.

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Zhang, X., Xi, Z., Zhao, H. et al. Efficient heterologous expression of bovine lactoferrin in Pichia pastoris and characterization of antibacterial activity. Syst Microbiol and Biomanuf (2024). https://doi.org/10.1007/s43393-024-00266-8

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  • DOI: https://doi.org/10.1007/s43393-024-00266-8

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