Ethanol-Inducible Bioproduction of Human α-Lactalbumin in <i>Komagataella phaffii</i>

α-Lactalbumin (α-LA) is the most abundant whey protein in human milk. Microbially expressed α-LA serves as a potential additive in infant formula to improve the protein composition and amino acid profile, enhancing the deep simulation of human milk. <i>Komagataella phaffii</i> is widely recognized for its ability to achieve high-density fermentation and robust secretion of heterologous proteins, making it ideal for large-scale production with relatively simple fermentation conditions. At present, the expression of human α-LA in <i>K. phaffii</i> remains challenged by the potential toxicity of using methanol as an inducer and inefficient bioproduction. In this study, we first employed the ethanol-transcriptional signal amplification device system in <i>K. phaffii</i> to express human α-LA, achieving a titer of 7.39 mg·L^-1 in shake flask fermentation. Next, through hybrid optimization of the native α-factor signal peptide and multicopy integration of the target gene, the α-LA titer was further increased to 16.52 mg·L^-1 in the shake flask. Finally, by addressing acetic acid accumulation in bioreactor fermentation, the engineered production strain achieved a titer of 0.60 g·L^-1 in a 3 L bioreactor. This work represents the first demonstration of high-efficiency methanol-free production of human α-LA in <i>K. phaffii</i> and provides strategies for the efficient expression and secretion of recombinant proteins in this host organism.