Production of phosphorylated and functional αs1-casein in Escherichia coli.

While the demand for milk proteins is on the rise, sustainability concerns dictate a reduction in animal-based agriculture. Microbial cell factories can be a viable alternative, but their inability to phosphorylate recombinant caseins is a major bottleneck, since phosphorylation is needed for calcium binding and functionality. We propose a solution that involves engineering bacteria to co-express bacterial protein kinases, resulting in recombinant caseins with the native phosphorylation pattern and functional properties. We successfully phosphorylated α_s1-casein in Escherichia coli using bacterial kinases, achieving phosphorylation at all native sites. To complement this approach, we developed an alternative phosphomimetic strategy by substituting serine residues with aspartate to mimic phosphorylation. Structural and functional characterization of all the phosphorylated/phosphomimetic recombinant α_s1-caseins demonstrated properties comparable with those of bovine α-casein, including calcium-binding affinity, digestibility, and structural integrity. Notably, phosphomimetic α_s1-casein may offer a simpler system, while phosphorylated casein more closely resembles bovine casein. This approach has significant implications for the development of alternative protein sources, addressing both sustainability and functional demands in the food industry.