High folate soybean

Symptoms of Folate Deficiency and the Deficiency Situation

Folate deficiency is directly related to neural tube defects (NTDs), megaloblastic anemia, cleft lip and palate, depression, tumors and other diseases [1]. NTDs are a group of defects caused by incomplete closure of the neural tube during embryo development, including anencephaly, encephalocele, spina bifida, etc. They are one of the most common neonatal defect diseases. The incidence rate of NTDs worldwide is approximately 0.5 to 2 per thousand. China is a country with a high incidence of NTDs. Every year, approximately 100,000 children with NTDs are born, with an incidence rate as high as 2.74 per thousand [1]. The preventive effect of folate on NTDs has been regarded as one of the most exciting medical discoveries of the late 20th century. Megaloblastic anemia is a type of anemia caused by the disorder of deoxy-ribonucleic acid synthesis due to a lack of folate or vitamin B12, and it is more common in infants and pregnant women. A normally developing fetus requires a large amount of folate reserves in the mother's body. lf the folate reserves are exhausted during labor or in the early postpartum period, it can lead to megaloblastic anemia in both the fetus and the mother. After supplementing folate, this disease can be recovered and cured rapidly [1]. Cleft lip and palate is one of the most common congenital birth defects, especially in China, with an incidence rate as high as 1.82 per thousand. On average, about 40,000 to 50,000 children with cleft lip and palate are born each year. The cause of cleft lip and palate is still unclear. Facts have proved that supplementing folate in the early stage of pregnancy can prevent the birth of children with cleft lip and palate [1].

According to statistics from the World Health Organization (WHO), folate deficiency has become a serious public health problem worldwide, with a persistently high prevalence rate showing an upward trend. The latest research in 2025 shows that more than 54% of the global population has insufficient folate intake. Among them, affected by the monotonous diet structure, the folate deficiency rate in developing countries is generally higher than that in developed countries, with Africa and South Asia being particularly severe. The folate deficiency rate among pregnant women in developing countries is as high as 45%. The "2022 Report on Chinese Residents' Nutrition and Health Status" by the National Health Commission of China shows that the folate deficiency rate among people aged 60 and above reaches 28.7%, which is significantly higher than that among middle-aged and young people (15.3%).

Under normal circumstances, the folate requirement for normal people is 100-200 μg/d. The recommended amount by the World Health Organization is: 200 μg/d for adults, and 400 μg/d for pregnant women and lactating mothers. The United States has implemented a mandatory folate fortification policy since 1998. It is estimated that folate-fortified cereal products can provide an additional 138 μg/d of folate to the American population [2]. Since the implementation of the folate fortification policy in the United States, the national blood folate level has increased significantly, and the incidence of neural tube defects has decreased significantly. Therefore, many countries around the world have also followed the example of the United States by fortifying staple foods with folate. As of 2021, 68 countries worldwide have implemented policies to fortify staple foods with folate.

Natural Biosynthesis Process of Folate

The natural biosynthesis of folate in plants includes 4 main processes. First, chorismate is converted into amino deoxy chorismate (ADCS) in plastids, and then into para-aminobenzoic acid (pABA). Second, GTP is converted into dihydroneopterin triphosphate (DHNTP) in the cytoplasm, and then into 6-Hydroxymethyl-7,8-dihydropterin (HMDHP). Then, pABA and HMDHP are sequentially reduced to dihydrofolic acid and tetrahydrofolic acid in mitochondria, and further form polyglutamyl tetrahydrofolic acid. Tetrahydrofolic acid is modified into different forms of folic acid in plants, such as 5-formyl-tetrahydrofolic acid, 10-formyl-tetrahydrofolic acid, 5,10-methenyl-tetrahydrofolic acid, 5,10-methylene-tetrahydrofolic acid, and 5-methyl-tetrahydrofolic acid. Among them, 5-methyl-tetrahydrofolate is the main form of active folate in the body [3].

The Shortcomings of Industrial Synthesis of Folic Acid

The traditional method for synthesizing folic acid is to use nitrobenzoic acid, and finally obtain folic acid through four steps: acylation, condensation, reduction and cyclization. Although this method can synthesize folic acid, the synthesis route is complicated, the production time is long, the production cost is high, and the yield is low, which is not conducive to large-scale production.

At present, folic acid is mainly synthesized in China using trichloroacetone, 2,4,5-triamino-6-hydroxypyrimidine sulfate and p-aminobenzoylglutamic acid as raw materials. Trichloroacetone, L-N-p-aminobenzoylglutamic acid, and 6-hydroxy-2,4,5-triaminopyrimidine sulfate are reacted for 5 hours at a reaction temperature of 40-45°C with the pH maintained at 3.0-3.5 in the presence of sodium metabisulfite and sodium carbonate to obtain folic acid. This operation process is simple, with short reaction time, easy control of conditions and low production cost. However, it has the disadvantage of large amounts of wastewater and waste gas. In the process of producing trichloroacetone, there are also serious wastewater pollution and serious waste gas pollution.

The Process of Folate Absorption in Humans

After the human body ingests dietary natural folate, it needs to hydrolyze polyglutamic acid into monoglutamic acid in the intestinal tract, which is then absorbed through active transport across the intestinal mucosa. Before entering the bloodstream, the monoglutamic acid form is reduced to tetrahydrofolate (THF) [4]. THF is further activated to 5,10-methylenetetrahydrofolate (5,10-MTHF), which is converted into 6S-5-MTHF that can be directly absorbed and utilized by the human body under the action of MTHFR [5]. Abnormalities in the MTHFR gene can affect the bioavailability of natural folate. 6S-5-MTHF transfers a methyl group to homocysteine (HCY) through the action of vitamin B12-dependent methionine synthetase (MTR), generating THF and methionine. Methionine is further converted into S-adenosyl methionine (SAM) [6]. SAM is an important substance in processes such as deoxyribonucleic acid (DNA) methylation and protein synthesis. In addition, 6S-5-MTHF is also involved in the synthesis process from deoxyuridine monophosphate (dUMP) to thymidylic acid (dTMP), and the deficiency of 6S-5-MTHF will lead to the obstruction of dTMP synthesis. Therefore, 6S-5-MTHF is an important substance for nucleic acid synthesis and DNA repair, and folate metabolism disorders will affect DNA methylation levels and interfere with DNA synthesis [7].

Therefore, compared with industrially synthesized folic acid, natural plant folate is more easily absorbed by the human body, and may increase absorption efficiency by directly providing 5-methyl-tetrahydrofolate, thereby overcoming the difficulty in folate absorption caused by MTHFR gene defects .

Different Plants Provide Different Contents of Folate

Folate is widely present in various plants, mainly including green leafy vegetables (such as spinach, amaranth), fruits (such as citrus fruits, kiwifruits), as well as beans and nuts (such as soybeans, chickpeas). These plants are important natural sources of folate for the human body. However, the content of folate varies greatly among different plants. For example, spinach (about 190-210 micrograms per 100 grams), amaranth (about 420 micrograms per 100 grams), citrus fruits (oranges, grapefruits, about 30-50 micrograms per 100 grams), kiwifruits (about 25-40 micrograms per 100 grams), soybeans, black beans, chickpeas (about 180 micrograms per 100 grams), peanuts, walnuts (about 50-100 micrograms per 100 grams).

Although these foods are rich in folate, due to the instability of the physicochemical structure of folate, it is easily decomposed, and the loss during food processing is very obvious, which also leads to the fact that the amount of folate ingested by the human body through diet cannot fully meet the daily needs. For example, the effective folate content in vegetables is easily reduced during heating and cooking, and the way of eating vegetables is relatively single with low daily demand. Although some vegetables have a high folate content, they still cannot meet the daily needs of the human body.

We Propose a Scheme for Designing High-folate Soybeans

Compared with vegetables and fruits, legume foods, on the one hand, have a higher level of folate content, and on the other hand, they are essential daily ingredients with diverse eating methods. They can be directly consumed as staple foods, made into various soy products including yuba, tofu, etc., and even made into various condiments including soy sauce, broad bean paste, etc. In addition, legumes are rich in active folate components, which can provide a more efficient way of folate supplementation for people with genetic defects in folate intake. Therefore, folate enhancement in legumes is an effective way to maintain human folate intake.

Based on the above background, we propose the concept of designing high folate soybeans by means of synthetic biology. The aim of dietary folate supplementation is achieved by enhancing the expression levels of key enzymes in the folate synthesis pathway in soybeans to increase the final folate synthesis amount. In our project, we overexpressed 4 folate synthases in soybean seeds, and in the obtained transgenic soybean plants, a significant increase in the content of active folate was detected. Our expected goal was successfully achieving in this project.

The In-depth Development and Extension of Our Project

Throughout the implementation of the entire project, we have developed new models to assist and advance the project, and have conducted a large amount of human practice and education work, providing support for the promotion of the project's core concepts and the practice of the integration of industry, academia and research.