Recombinant Production of Bovine α-Casein in Genome-Reduced Strain IIG-Bs-20-5-1.

Background: Cow's milk represents an important protein source. Here, especially casein proteins are important components, which might be a promising source of alternative protein production by microbial expression systems. Nevertheless, caseins are difficult-to-produce proteins, making heterologous production challenging. However, the potential of genome-reduced was applied for the recombinant production of bovine α-casein protein.

Methods: A plasmid-based gene expression system was established in allowing the production of his-tagged codon-optimized bovine α-casein. Upscaling in a fed-batch bioreactor system for high cell-density fermentation processes allowed for efficient recombinant α-casein production. After increasing the molecular abundance of the recombinant α-casein protein using immobilized metal affinity chromatography, zeta potential and particle size distribution were determined in comparison to native bovine α-casein.

Results: Non-sporulating strain BMV9 and genome-reduced strain IIG-Bs-20-5-1 were applied for recombinant α-casein production. Casein was detectable only in the insoluble protein fraction of the genome-reduced strain. Subsequent high cell-density fed-batch bioreactor cultivations using strain IIG-Bs-20-5-1 resulted in a volumetric casein titer of 56.9 mg/L and a yield of 1.6 mg/g after reducing the protein content. Comparative analyses of zeta potential and particle size between pre-cleaned recombinant and native α-casein showed pH-mediated differences in aggregation behavior.

Conclusions: The study demonstrates the potential of for the recombinant production of bovine α-casein and underlines the potential of genome reduction for the bioproduction of difficult-to-produce proteins.