Nowadays, many mothers are unable to breastfeed exclusively for various reasons and have to choose to use formula as a substitute or supplement. However, the baby formula on the market cannot fully meet the needs of infants and toddlers as there is still a gap between the composition of cow’s milk-based formula and breast milk. And formula with comprehensive nutrition is usually more expensive, which puts financial pressure on many families. Therefore, we chose to use Saccharomyces cerevisiae to synthesize lactoferrin and N-acetylneuramin, which are essential for infant growth, as an additional supplement to the formula. We wish to alleviate the financial burden of many families through our projects and promote the healthy growth and intellectual development of infants and children.

As we all know, infancy and early childhood are critical stages in human development. Paying attention to the healthy growth of infants and young children is not only related to the happiness of every family but also has profound significance for the development of the entire society. The human body and brain develop rapidly during infancy and early childhood, and good nutrition is crucial for growth at this stage.

For infants and young children, breast milk is the most ideal food, and the proportion of its nutritional components can meet the needs of babies at different growth stages. Breast milk is not only easy to digest and absorb, but also the immunoglobulin, lactoferrin, and other immune factors rich in it can build a solid health defense line for babies and help them resist the invasion of various diseases. However, many mothers cannot achieve full breastfeeding for various reasons. In addition to physical health problems caused by the COVID-19 epidemic and life pressure, an important factor is that many working mothers no longer have the condition of direct breastfeeding after returning to work. In 2023, the number of newborns in China is 9.02 million, but the exclusive breastfeeding rate of infants under 6 months is only 36.2%, and the breastfeeding rate after 12 months is only 46%. Therefore, many mothers have to use infant formula as a substitute and supplement.

Data source: 《White Paper on Infant and Young Child Feeding in China 2023》

However, there is an obvious gap in nutritional composition between formula milk powder on the market and breast milk. Although they are constantly improving, they still cannot fully simulate the complexity and superiority of breast milk. The rich nutritional components in breast milk are crucial for the development of the immune system, growth, and health of infants and young children, and these components are difficult to be completely replicated in formula milk. This has left more than 76% of mothers concerned that their children are not getting enough nutrients, which could affect their healthy development.

At the same time, formula with more comprehensive nutrition is often very expensive, which puts a huge economic pressure on many families. More than 60% of families spend more than $1,680 on formula milk powder every year, while the per capita disposable income of the country is only $5,491.

Data source: "https://www.naifenzhiku.com/article/cate-7/detail-6496.html"

For some families, the cost of buying formula milk powder accounts for a large part of family expenses, bringing a burden to family economy and causing restrictions on their expenditures in other aspects and affecting the quality of family life. This phenomenon makes working mothers from middle and low-income families fall into a dilemma: breastfeeding is difficult to balance work and life, which may make family income lower; but formula feeding is difficult to afford and may make their children lack nutrition.

Having learnt about the above problems, we were very anxious and eager to do something to alleviate the pressure on these families. We wondered if it would be possible to develop a product that could be used as a supplement to formula milk to provide additional nutrition for infants and young children. Such a product should be inexpensive so that more families can have access to it.

After conducting questionnaires and reviewing the literature, we found that two substances, lactoferrin and N-acetylneuraminic acid, are essential for the growth and development of infants and children.

N-acetylneuraminic acid, abbreviated as Neu5Ac, is a crucial component of infant brain gangliosides. During early childhood, Neu5Ac plays a vital role by regulating the growth of nerve cells and influencing the transmission of nerve impulses in neurons, thereby expediting the intellectual development of infants. Due to its unique structure, N-acetylneuraminic acid possesses many physiological and biochemical functions important to human health. Thus, it significantly contributes to brain development during infancy and throughout childhood.

However, it has been reported that the N-acetylneuraminic acid content in breast milk decreases over time. Furthermore, formula milk contains only 15-35 mg/litre of Neu5Ac, which is far from sufficient for the daily needs of infants and young children.

Lactoferrin is a multifunctional protein found in high levels in human colostrum and lower levels in mature breast milk. Adding lactoferrin to infant supplements provides multiple benefits, especially in protecting infants and young children from bacterial, viral, and fungal infections. Lactoferrin achieves this by binding with iron, making it unavailable to bacteria.

The content of lactoferrin in breast milk is approximately 2.5g/L, while most baby formulas contain only 0.05g/L. A lack of lactoferrin during feeding may impair an infant’s intestinal barrier and innate immune functions, disturb the intestinal microbiota, and result in neuro-immune dysfunction and developmental delays in the hippocampus.

Therefore, for infants who rely solely on powdered milk, lactoferrin supplementation is particularly crucial during this critical period of growth and development, as sufficient nutrients are required to support bodily development.

Based on the above information, our team developed an engineered Saccharomyces cerevisiae that produces lactoferrin and N-acetylneuraminic acid as a nutritional supplement for infants and young children, helping the next generation grow healthily and develop intellectually.

We chose Saccharomyces cerevisiae as the chassis cell for the production of lactoferrin and N-acetylneuraminic acid not only because it is a well-researched model organism, but also because yeast is widely used in the food industry and its safety can be guaranteed.

In our experiments, we selected the recombinant vector pESC-URA containing GAL1 and GAL10 promoters to express our genes and ensure stable gene expression. The GAL1,10 promoters are commonly used for expression in yeast and broadly in eukaryotes. They are one of the promoters that have been well-studied in all eukaryotic organisms, and can be activated in the presence of galactose and the absence of glucose.

The control of these repressible promoters allows one or two cloned genes to be introduced into a yeast host strain with the pESC-URA vector. This vector enabled us to achieve the biosynthesis of heterologous proteins, leading to the synthesis of both lactoferrin and N-acetylneuraminic acid.

We chose to introduce a human-derived lactoferrin synthesis gene into the engineered yeast to complete the lactoferrin synthesis process. Since the lactoferrin encoded by the human gene is structurally and functionally identical to natural lactoferrin in the human body, it provides higher nutritional value and health benefits.

For N-acetylneuraminic acid, after conducting a literature review, we selected the most efficient synthetic genes known to result in higher yields, ensuring optimal production in the engineered yeast.

1. 刘爽, 陈磊, PHILIP HASELBERGER, 等. 离子色谱法测定母乳和牛乳中的N-乙酰神经氨酸[J]. 中国食品学报, 2021, 21(9): 250-256. DOI: 10.16429/j.1009-7848.2021.09.027.

2. 卢晓婷, 刘钊燕, 罗贇, 等. N-乙酰神经氨酸对婴幼儿的营养功能及其在婴幼儿配方产品中的应用现状[J]. 现代预防医学, 2020, 47(16): 2953-2957.

3. Hassan M I, Lundgren B R, Chaumun M, et al. Total biosynthesis of legionaminic acid, a bacterial sialic acid analogue[J]. Angewandte Chemie, 2016, 128(39): 12197-12200.

4. Röhrig C H, Choi S S H, Baldwin N. The nutritional role of free sialic acid, a human milk monosaccharide, and its application as a functional food ingredient[J]. Critical Reviews in Food Science and Nutrition, 2017, 57(5): 1017-1038.

5. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), Turck D, Bresson J L, et al. Safety of synthetic N‐acetyl‐d‐neuraminic acid as a novel food pursuant to Regulation (EC) No 258/97[J]. EFSA Journal, 2017, 15(7): e04918.

6. 刘彦品, 董学艳, 姜铁民, 等. 母乳中乳铁蛋白含量及影响因素研究[J]. 中国食品添加剂, 2018(8): 70-74. DOI: 10.3969/j.issn.1006-2513.2018.08.005.

7. Giansanti F, Panella G, Leboffe L, et al. Lactoferrin from milk: Nutraceutical and pharmacological properties[J]. Pharmaceuticals, 2016, 9(4): 61.

8. Manzoni P, Dall'Agnola A, Tomé D, et al. Role of lactoferrin in neonates and infants: an update[J]. American journal of perinatology, 2018, 35(06): 561-565.

9. Wang W, Cheng Z, Wang X, et al. Lactoferrin deficiency during lactation increases the risk of depressive-like behavior in adult mice[J]. BMC biology, 2023, 21(1): 242.

10. Jańczuk A, Brodziak A, Czernecki T, et al. Lactoferrin—The Health-Promoting Properties and Contemporary Application with Genetic Aspects[J]. Foods, 2022, 12(1): 70.

11. Kell D B, Heyden E L, Pretorius E. The biology of lactoferrin, an iron-binding protein that can help defend against viruses and bacteria[J]. Frontiers in immunology, 2020, 11: 550441.

12. https://www.snapgene.com/plasmids/yeast_plasmids/pESC-URA

13. https://www.agilent.com/cs/library/usermanuals/Public/217451.pdf

14. Elison G L, Xue Y, Song R, et al. Insights into bidirectional gene expression control using the canonical GAL1/GAL10 promoter[J]. Cell reports, 2018, 25(3): 737-748. e4.

15. Conesa C, Calvo M, Sánchez L. Recombinant human lactoferrin: a valuable protein for pharmaceutical products and functional foods[J]. Biotechnology Advances, 2010, 28(6): 831-838.

16. Cazacu A L. Lactoferrin: analysis of the structure profile[J]. Chemistry Journal of Moldova, 2014, 9(2): 99-106.