Transplastomic biofactory for the production of functional human α-lactalbumin for nutritional and therapeutic applications.

The sustainable production of functional human proteins in plants offers a transformative path for developing animal-free nutritional and therapeutic compounds. In this study, we report one of the first expression of human α-lactalbumin (hLA), a key milk protein with nutritional and anticancer properties, via chloroplast genome engineering in Nicotiana tabacum. A codon-optimized hLA gene was introduced into the plastid genome using a synthetic expression cassette under the control of strong plastid regulatory elements. Homoplasmic transplastomic lines were obtained and confirmed via PCR and Southern blot analysis. Immunoblotting and ELISA quantification revealed that hLA accumulated to 23.4% of total soluble protein (TSP), one of the highest levels reported for plastid-expressed human proteins. Far-UV circular dichroism (CD) spectroscopy confirmed that the chloroplast-derived hLA adopted a native-like α-helical structure. Functionally, the recombinant protein successfully activated galactosyltransferase (GalT) in vitro, enabling lactose synthesis at 93% of the rate observed with native hLA. Furthermore, chloroplast-derived hLA was converted into a bioactive HAMLET complex by combining with oleic acid under mild thermal conditions. This complex induced potent apoptosis in human colorectal (WiDr) and breast cancer (MCF-7) cells, reducing viability to less than 8%, as confirmed by MTT and Annexin V/PI assays. These findings establish chloroplasts as an effective platform for high-yield, correctly folded, and functional production of human milk proteins. The ability to generate both enzymatically active and therapeutically functional products from a single plant-based system underscores its potential in food engineering and synthetic biology. This work offers a scalable, sustainable, and dual-purpose strategy for the development of recombinant milk proteins applicable in infant nutrition, functional foods, and plant-made biotherapeutics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-38965-y.