Engineering a transport pathway to boost extracellular production of human lactoferrin in Komagataella phaffii

Human lactoferrin (HLF), a pivotal iron-binding glycoprotein belonging to the transferrin family, is the primary immune protein in breast milk. Currently, recombinant HLF expression in microorganisms has attracted widespread attention. Through genome integration and copy number optimization, this study established a Komagataella phaffii cell factory that achieved an HLF titer of 137.6 mg/L. However, HLF accumulated extensively within the cells. Subsequently, several endogenous signal peptides were screened and hybridized with mating factor α, resulting in a 1.56-fold increase in extracellular HLF titers compared to the control. Additionally, protein kinase II overexpression facilitated vesicle trafficking, further increasing extracellular HLF to a titer of 304.6 mg/L in shake flasks and 1.7 g/L in a 3-L bioreactor. Notably, the intracellular/extracellular HLF concentration ratio decreased from 1.69:1 to 1.01:1. Further enhancements were made through ion optimization and an exponential methanol-feeding strategy, resulting in an extracellular HLF titer of 3.1 g/L. To the best of our knowledge, this is the first study to report considerably improved extracellular HLF production through optimization of the transport and secretion pathways and provide insights into the expression of other secretary human proteins in K. phaffii.