The global demand for milk comes from the fact that it is one of the best sources of protein because of its complete amino acid profile. However, because milk comes from animal-based agriculture, its production has significant environmental impacts and ethical considerations. Researchers have provided a workaround to producing the milk protein casein using bioengineered Escherichia coli bacteria. Details of their study are discussed in a July 2025 paper published in the journal Trends in Biotechnology.
Replicating Casein for Sustainable Dairy Alternatives via Engineered E. Coli: Solving the Phosphorylation Puzzle is Key to Lab-Grown Casein
Background
Casein is essential in human nutrition due to its complete amino acid profile and digestibility. This protein was worth about USD 2.7 billion in 2023. But traditional production relies on animal farming. Microbial cell factories have been positioned as an alternative to rearing cows for milk production, but achieving the correct phosphorylation pattern is the biggest hurdle.
Phosphorylation is a process where phosphate groups are added to proteins. For casein, this modification is critical for its ability to bind calcium, which in turn is essential for forming casein micelles. Casein micelles are tiny structures found in milk. They are responsible for many of the functional properties of milk, like stability, texture, and nutritional value.
It is also important to underscore the fact that microbe-produced casein will not behave like real milk protein in food products like cheese and yogurt without proper calcium binding and micelle formation. Researchers S. Balasubramanian et al. took on this scientific challenge. Their goal was to create a protein that is functionally and nutritionally comparable to bovine casein.
Strategies
The researchers successfully demonstrated two key strategies to overcome the phosphorylation challenge mentioned above:
1. Bacterial Kinase Expression: The first involved engineering Escherichia coli bacteria to express bacterial enzymes called kinases. These kinases are able to add phosphate groups to the recombinant αs1-casein. This mimics the natural phosphorylation pattern found in milk from cows.
2. Phosphomimic Approach: This is another alternative approach where, instead of actually phosphorylating the protein, the researchers made genetic modifications to substitute certain serine amino acids with aspartate. The negative charge of aspartate mimics the effect of phosphorylation.
Both the phosphorylated and phosphomimic caseins were tested for their ability to bind calcium. This is critical in the initial stage. Structural and functional tests like calcium-binding affinity, digestibility, and structural integrity showed that both phosphorylated and phosphomimetic recombinant caseins had properties comparable to those of bovine casein.
The kinase-mediated method offers a closer resemblance to natural casein. The phosphomimetic technique provides a more efficient production process. Researchers emphasize the importance of further studies to optimize yield and validate long-term performance. These approaches mark a significant step toward scalable and animal-free milk protein production.
Takeaways
The solutions have been successfully demonstrated at the laboratory scale. However, because the lab is a controlled environment, there is a need to translate the processes in large-scale production. An assessment showed that the Technology Readiness Level is at TRL 4. This indicates significant progress, but more development is needed before commercialization.
Nevertheless, because of the billion-dollar value of casein in the global market, the implications of the study are immense. It offers a path forward for the food industry to develop authentic-tasting and nutritious dairy alternatives that do not require livestock. This reduces greenhouse emissions and promotes ethical food production and consumption practices.
It is also worth mentioning that the study demonstrates an approach called precision fermentation, which involves manufacturing protein using microbes or bacteria. Some bacteria can be engineered to express specific proteins that are as complex as animal proteins. Microbes also have incredibly short doubling times. This allows for rapid and large-scale production.
FURTHER READING AND REFERENCE
- Balasubramanian, S., Mobasseri, G., Shi, L., Jers, C., Køhler, J. B., Boire, A., Berton-Carabin, C., Mijakovic, I., and Jensen, P. R. 2025. “Production of Phosphorylated and Functional αs1-Casein in Escherichia coli.” Trends in Biotechnology. DOI: 1016/j.tibtech.2025.05.015