Precision fermentation of milk proteins

By 2050, the world’s population is expected to reach 9.1 billion, driving a significant increase in global demand for animal-based proteins. This demand is fueled by higher living standards and shifting dietary preferences toward protein-rich foods. While animal-based proteins provide essential nutrition, their production has a substantial environmental impact. To address this challenge, there is an urgent need for sustainable alternatives that are nutritionally equivalent, functionally superior, and environmentally friendly.
Microbial production systems, particularly precision fermentation, offer a promising solution for producing high-quality mammalian proteins sustainably. However, several challenges must be addressed, including low yields, high production costs, the absence of post-translational modifications, and a limited understanding of the structural and functional properties of recombinant proteins produced in microbes. Overcoming these bottlenecks is essential to fully realize the potential of precision fermentation in delivering sustainable, high-quality protein solutions for the growing global population.
This thesis delves into precision fermentation, focusing on the production of milk proteins using bacterial strains Escherichia coli (E. coli) and Corynebacterium glutamicum (C. glutamicum). It explores how these microbial systems can be leveraged to produce animal-free milk proteins efficiently, paving the way for sustainable solutions in the food industry.
Chapters II and III examines the potential of C. glutamicum as a host for recombinant milk protein production. The study begins by mapping the host’s secretome to identify all proteins present in the culture medium. This comprehensive mapping allows researchers to pinpoint efficient genetic elements—such as promoters, signal peptides, and proteases - that enhance protein expression and secretion. Simultaneously, the mapping process identifies unnecessary proteins that could be knocked out to streamline the system. Using this information, a promoter and signal peptide were selected and tested for their efficiency in secreting heterologous whey proteins. The results demonstrated that the chosen genetic elements facilitated efficient secretion, achieving higher levels of the target proteins compared to previous construct.
The focus then shifts to E. coli, a well-established model organism in recombinant protein production. In this context, the chapters IV - V highlights experiments involving the expression of α_s1- and β-casein in E. coli and the subsequent testing of various bacterial and mammalian kinases. Remarkably, bacterial kinases were found to phosphorylate milk proteins efficiently, achieving functional characteristics similar to those observed in natural bovine proteins. Additionally, an alternative approach employing phosphomimetic mutations was introduced. This strategy substitutes phosphorylation by replacing specific amino acids with negatively charged residues, mimicking the structural and functional effects of phosphorylation.
The structural and functional characterization of these recombinant caseins revealed that both phosphorylated and phosphomimetic caseins, exhibited properties comparable to their bovine counterparts. For instance, calcium-binding assays and digestion studies demonstrated that the recombinant caseins mirrored the mineral-binding capacity and digestibility of natural bovine caseins, confirming their potential for use in various food applications.
Lastly, chapter VI explores the application of these recombinant caseins in plant-animal protein hybrids, a growing area of interest in sustainable food development. By testing the interactions and associative behavior of recombinant milk proteins with plant proteins such as napin and legumin, the study revealed promising results. The recombinant proteins exhibited behavior comparable to that of bovine milk proteins, supporting their potential integration into hybrid formulations for improved texture, stability, and functionality in food products.
In summary, this thesis offers a comprehensive exploration of precision fermentation as a promising platform for milk protein production. From optimizing genetic elements in C. glutamicum to developing phosphorylated and phosphomimetic proteins in E. coli, it demonstrates how microbial hosts can produce milk proteins with structural and functional characteristics similar to bovine counterparts. Additionally, the findings underscore the applicability of these proteins in hybrid plant-animal systems, presenting a promising avenue for sustainable food innovation.