Secretory expression of porcine lactoferrin in Trichoderma reesei and characterization of the recombinant protein

Abstract

Lactoferrin exerts a variety of physiological functions, including iron metabolism regulation, antibacterial activity and antiviral activity, indicating its considerable application potential in the feeding of weaned young animals. As porcine lactoferrin (PLF) is scarce in source, bovine lactoferrin and other substitutes are generally utilized in related studies on piglet feeding. Trichoderma reesei possesses efficient protein secretion ability and eukaryotic post-translational modification capacity, and has been successfully used for the efficient expression of various food and feed enzyme preparations. This study aimed to construct an engineered T. reesei strain capable of efficiently expressing porcine lactoferrin (PLF), and to conduct a preliminary analysis of the recombinant protein. Using T. reesei RUT-C30Δpyr4Δtku70 as the parental strain, the protease activation factor gene (pea1) and cellobiohydrolase II gene (cbh2) were sequentially knocked out via CRISPR/Cas9 technology, resulting in the construction of recombinant expression host strains C30Δpyr4Δtku70Δpea1 and C30Δpyr4Δtku70Δpea1Δcbh2, respectively. In these constructed host strains, the expression of the porcine lactoferrin gene (plf) was driven by the strong inducible promoter P_cbh1. Shake-flask fermentation assays demonstrated that knockout of the pea1 gene significantly enhanced the yield and stability of PLF in the fermentation broth. In a 30-L fermenter, the recombinant strain C30Δpyr4Δtku70Δpea1Δcbh2Δcbh1::plf achieved a maximum PLF yield of 2.37 g/L. The purified PLF protein was also subjected to iron-removal and iron-binding assays, with evident color changes observed. Iron saturation analysis confirmed that PLF could bind iron ions in iron-rich solutions, thereby increasing its iron saturation. In contrast, in iron-removal solutions, iron-saturated PLF released iron ions, leading to a reduction in the protein’s iron saturation level. This study is the first to achieve the heterologous expression of PLF in T. reesei. The recombinant PLF exhibited intact iron-binding and iron-release functions, laying a key foundation for further investigations into its application in piglet nutrition.

Keywords

Trichoderma reesei / Recombinant porcine lactoferrin / Protease-activating factor / Iron saturation

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Funding

National Key Research and Development Program of China(2022YFC2104602)

RIGHTS & PERMISSIONS

Jiangnan University