Abstract:Human lactoferrin is involved in various biological activities, such as antimicrobial, anti-inflammatory, and anti-tumor activities, as well as immune regulation. Thus, it has broad application prospects in food, cosmetics, and feed additives. However, the high cost of extracting lactoferrin from milk restricts its mass production and application. A potential solution is to produce recombinant human lactoferrin (rhLF) by constructing a high-yield cell factory. In this study, the promoter and signal peptide in the rhLF gene expression element were optimized, resulting in an almost four-fold increase (from 215.00 to 849.52 mg/L) in total protein expression in the supernatant. Furthermore, rhLF production was increased by overexpressing protein secretion-promoting factors related to the rhLF production and secretion pathway and regulating cellular stress response. After the redox state of yeast cells under production pressure was balanced by overexpressing Yap1 (which regulates the glutathione redox system) and Msn2 (which enhances oxidative tolerance), the total protein in the supernatant of rhLF-expressing strains increased from 849.52 mg/L to 1 055.68 mg/L. Finally, the target protein was semiquantified, showing an almost 4.8-fold increase (from 34.82 mg/L to 197.30 mg/L). In this study, the protein production capacity of rhLF-expressing strains was greatly improved by combining various strategies, providing guidance for the efficient secretory expression of rhLF in Pichia pastoris.
