Multi-modular engineering of Komagataella phaffii for efficient synthesis of human lactoferrin

Human lactoferrin is widely used in medical and nutritional fields. However, current production methods, which rely heavily on extraction from bovine or ovine milk, suffer from long production cycles, high costs, and structural and functional differences from the human protein. To address these challenges, this study constructed a Komagataella phaffii (K. phaffii) cell factory using methanol as the sole carbon source, aiming to develop an efficient and sustainable biosynthetic route for human lactoferrin. During the expression design phase, a novel configuration formed by the "CAAAAC" Kozak sequence and the PHO11α signal peptide achieved a secreted yield of 26.1 mg/L (strain K1). Building on this, multi-copy integration using rDNA non-transcribed regions increased the yield to 60.5 mg/L, but also revealed a production bottleneck associated with simply increasing copy number. To address this, fluorescence protein monitoring technology was introduced to precisely dissect the bottleneck, guiding subsequent multi-layer metabolic engineering that further increased the yield to 90.3 mg/L. Critically, AFT1 and SEC12P were identified for the first time in K. phaffii as novel positive regulatory targets. Additionally, morphological engineering confirmed that deletion of the PHA1 gene synergistically enhanced protein accumulation. Ultimately, the engineered strain integrating these multi-layer strategies achieved a human lactoferrin titer of 690 mg/L in 3-L fed-batch fermentation, a leading level among reported heterologous synthesis systems. This study not only provides a new efficient route for human lactoferrin production but also offers new insights for the advanced engineering of K. phaffii as a protein synthesis factory.