Harnessing Yarrowia lipolytica as a platform for animal-free and secretory production of human lactoferrin.

Human lactoferrin (hLF) is an iron-binding glycoprotein with important nutritional and therapeutic functions. Microbial production of recombinant hLF offers a scalable and animal-free alternative to extraction from milk, but efficient secretion remains a major challenge. Here, we developed Yarrowia lipolytica as a microbial platform for high-level production and secretion of recombinant hLF. By systematically improving protein expression, secretion, and protein-folding factors, we identified a key limitation in hLF production: simply increasing gene copy number did not proportionally increase extracellular hLF yield and instead caused impaired cell growth and morphology. We therefore enhanced the cellular protein-folding and secretory machinery, which substantially enhanced the cells to accommodate and secrete higher levels of hLF. Through this coordinated engineering strategy, the best-performing strain produced 409.9 mg/L secreted hLF in shake flasks. Further process optimization in a 5-L fed-batch fermentation increased the hLF titer to 3.14 g/L. These results demonstrate that balancing recombinant protein expression with host secretory capacity is critical for efficient production of complex proteins. More broadly, this work establishes Y. lipolytica as a promising platform for sustainable, animal-free manufacturing of lactoferrin and potentially other high-value nutritional and therapeutic proteins.