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

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.

Funding

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)

the Program of Introducing Talents of Discipline to Universities,(111-2-06)

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