Milk protein produced in E. coli using fermentation

New techniques are bringing alternative dairy proteins closer to commercial use. Researchers have successfully produced functional, phosphorylated αs1-casein in Escherichia coli—an important step toward sustainable protein production through precision fermentation.

Phosphate groups crucial for binding

Casein only becomes functional once it can effectively bind calcium. This binding is essential for the formation of micelles in milk, which plays a key role in products like cheese. But phosphorylation is required for this function—something bacteria such as E. coli don’t naturally perform.

The study tested two methods: co-expression of bacterial kinases PrkD and YabT, and a so-called phosphomimetic approach, in which phosphorylation sites are replaced with negatively charged amino acids. Both approaches resulted in functional proteins. “PrkD phosphorylated all nine native sites, and YabT phosphorylated eight of them,” the team reported. The phosphorylation was confirmed through mass spectrometry and 2D gel electrophoresis.

Functional properties under review

The casein variants were tested for digestibility, calcium-binding capacity, and structural properties. All variants were fully broken down in simulated digestive fluids. Both the phosphorylated and phosphomimetic versions showed a clear increase in calcium affinity compared to the non-phosphorylated counterpart.

Fluorescence spectroscopy revealed that the protein’s structure changed as a result of the modifications. The results suggest minor shifts, likely caused by changes in charge and interactions with water.

Industrial potential on the horizon

The researchers estimate the Technology Readiness Level of this approach at 4. Large-scale production is not yet feasible, but the path is becoming clearer. Previous studies have shown that E. coli can achieve high protein yields. Other microorganisms, such as Bacillus or Saccharomyces, may also offer more efficient production and secretion of this type of casein.