Recombinant Expression and Biosynthesis of Milk Proteins
Natural animal milk and its dairy products are the most ideal sources of protein nutrition, with their nutritional components primarily being casein and whey proteins, as well as containing bioactive peptides, fats, lactose, vitamins, and minerals. However, on the other hand, natural animal milk, due to containing various allergens, ranks as the top allergenic food among infants and young children. Additionally, milk contains about 4.6% lactose, leading to adverse symptoms such as bloating and diarrhea in lactose-intolerant individuals. Especially in recent years, animal milk production has been facing issues such as high breeding costs, shortage of feed resources, antibiotic overuse, severe environmental pollution, and challenging disease prevention and control situations.
Therefore, alternative protein technologies for dairy products have garnered widespread attention. In recent years, advancements in biotechnologies such as gene editing, synthetic biology, and cellular agriculture have provided transformative production methods for global food production, with laboratory-made products such as cultured meat, artificial eggs, and animal-free milk continuously emerging. Using synthetic biology techniques, specific synthetic capabilities can be engineered into cellular factories to produce various agricultural products such as starch, proteins, fats, sugars, milk, and meat needed by humans. For example, designing yeast cells for industrial fermentation of bovine milk proteins (such as whey protein and casein) and corresponding nutritional components can achieve green manufacturing of animal-free dairy products.
Biosynthesis of Milk Proteins
Milk proteins are primarily composed of casein and whey proteins, accounting for approximately 80% and 20% of the total protein, respectively. Whey contains mainly β-lactoglobulin, α-lactalbumin, lactoferrin, fat globule membrane proteins, and immunoglobulins. The expression of milk proteins is the core of animal-free milk biosynthesis, mainly referring to the efficient expression of various protein components from animal milk using genetic engineering and cellular factory technologies. Currently, research on animal-free milk is in its early stages of breakthroughs in production processes and commercialization of products, with technical bottlenecks awaiting breakthroughs in efficient synthesis of important milk protein components and production processes of synthetic dairy products.
Expression of milk proteins initially involved obtaining milk proteins through transgenic livestock milk. The obtained milk proteins are modified and processed to have stable bioactivity, which can be directly used in food processing. In the early 21st century, plant expression systems were constructed using plant tissue cells and chloroplasts to create plant expression systems for milk protein production. In the past decade, synthetic biology based on genomics and systems biology has made breakthrough progress in animal-free milk research by using a series of model microorganisms and plants as chassis cellular factories for efficient expression of milk proteins.
Recombinant Expression of Milk Proteins
Efficiently expressing various protein components from animal milk using genetic engineering and cellular factory technologies is a core technical aspect of animal-free milk biosynthesis. Additionally, combining the expression of milk proteins is a key technology for obtaining animal-free milk. For example, transgenic cows' mammary glands are engineered to express various milk proteins, producing food formulas containing multiple recombinant peptides. Using microbial cellular factories to recombinantly express milk proteins generates compounds with a flavor, appearance, nutritional value, aroma, and similar taste to bovine milk produced by mammals. Lactoferrin expression can also be added to feed and used as an ingredient in infant food formulas.
Microbial Synthesis of Animal-free Milk
The new process of animal-free milk biosynthesis involves using microbial fermentation to produce dairy protein. By artificially synthesizing the DNA sequences of bovine milk proteins and transforming them into food-grade yeast to express casein and whey proteins, animal-free milk without lactose, cholesterol, and allergens is produced by blending with plant-based ingredients. This method provides a composition without animal-derived components, containing κ-casein, β-casein, lipids, flavor compounds, and sweeteners. Animal-free milk mixes non-animal proteins such as β-lactoglobulin and pea protein, involving non-animal protein food and production, recombinant bovine milk protein, filamentous fungi cell culture, as well as casein formulations and manufacturing methods.
Microbial Cellular Factories for Milk Protein Expression
Early technologies utilized animal and plant bioreactors to express milk proteins and their combinations, enabling the direct obtainment of edible milk proteins. However, extraction, separation, and purification of milk proteins were difficult, and production costs were high. Modern techniques, such as synthetic biology, are utilized to transform natural milk proteins and their microbial expression hosts, with combined milk protein expression being a significant trend in current technical development. To achieve the bio-manufacture of alternative milk proteins, efficient expression of milk proteins in microbial host cells needs to be established.
- Yeast
Yeast is the most widely used platform for efficient expression of recombinant proteins, having extensive applications in industries such as agriculture, food, and medicine. Currently, the engineering of yeast chassis has gradually transitioned from version 1.0 of high-level expression of enzymes and proteins at the genetic level, version 2.0 of metabolic pathway regulation at the omics level, to the new stage of intelligent artificial design version 3.0. By developing modular expression systems, reconstructing genetic elements, and large-scale recombination and rearrangement of genes, optimization of protein synthesis pathways and metabolic regulation networks, as well as global transformation of chassis cells, create low-energy, high-yield, easily processable expression systems for the efficient production of recombinant bovine milk proteins. An article titled "Synthetic biology 2020–2030: six commercially-available products that are changing our world" was published in 2020, listing six biologically synthesized products that have been commercialized between 2000 and 2020 and are changing the world, including the use of recombinant yeast to express soy hemoglobin, as a future food ingredient to improve its taste and flavor.
- Filamentous Fungi
Filamentous fungi are more suitable as microbial hosts for producing recombinant mammalian proteins. Compared to yeast, filamentous fungi have a stronger capability for industrial production of recombinant proteins, and the recombinant proteins produced have secretion characteristics and post-translational modifications such as glycosylation more similar to those produced by mammalian cells. Progress has been made in the protein secretion expression systems and genetic manipulation systems of filamentous fungi, such as obtaining efficient protein expression hosts like Aspergillus niger and Trichoderma reesei through genetic modifications, exploring expression elements such as strong promoters, signal peptides, terminators, anchoring proteins, and transcription factors related to protein secretion regulation, constructing filamentous fungi protein secretion expression systems, and the core platform for industrial-scale efficient expression of recombinant exogenous proteins. Additionally, through optimizing large-scale cultivation and screening of filamentous fungi, efficient recombinant production of milk proteins will be applied to chassis cell factories.
Next-Generation Animal-free Milk Biomanufacturing
Compared to traditional milk production methods, animal-free milk biosynthesis processes have significant environmental advantages. Animal-free milk products have a notable advantage over natural animal milk products in that they do not contain adverse factors such as lactose, cholesterol, and allergens. By comparing and analyzing allergen databases, artificial design of different source milk protein sequences can enhance their bioactivity while removing allergens, potentially obtaining more nutritious and healthier animal-free milk products.
However, in terms of taste, flavor, and nutrition, current animal-free milk products cannot yet compete with naturally derived animal milk products. In natural cow's milk, the protein content is only about 3%, mainly composed of two major parts: casein and whey proteins, but its flavor and active ingredients are very complex. Unique flavor substances in milk include various organic compounds such as free fatty acids, alcohols, esters, lactones, aldehydes, ketones, phenols, ethers, sulfur-containing compounds, and terpenes. Among them, fat, as an important food component, is crucial for improving the flavor, taste, and nutritional quality of future synthetic dairy products. Therefore, utilizing synthetic biology techniques to transform microbial chassis, combining the synthesis of various lipid flavor substances and peptide bioactive substances, broadens the application prospects of animal-free milk.
Reference
- Voigt, C. A., Synthetic biology 2020–2030: six commercially-available products that are changing our world, Nature Communications, 2020, 11, 6379.
