Abstract 

According to the assessment of nutritional status in China, vitamin B2 deficiency is one of the biggest health threats to Chinese people, especially in some less-developed areas. Our project aims to solve this problem. We integrated the feedback from different stakeholders, including the general public, genetic engineering experts, potential customers, bakeries, and food industry experts into our project. And we improved and finalized our project to be more realistic and responsible. Our project included the following aspects: public education on nutritional supplementation, the use of engineered yeast to produce vitamin B2-enriched food, safety considerations, and specialized designs for different groups.

Introduction 

The release of the "National Nutrition Plan of China (2017-2030)" in 2017 drew our interest to the dietary structure and nutrition. We found that in the Chinese population, the problem of vitamin B2 deficiency is the most severe among all the vitamins.

Vitamin B2, also known as riboflavin, is involved in energy metabolism, cellular respiration, and antibody production, as well as normal growth and development. Vitamin B2 deficiency can lead to inflammation and organic disorders of the mouth, lips, skin, and genitalia (Xiang et al., 2004).

Foods that are rich in vitamin B2 are beef, pork, chicken, fish, and dairy products. People from less developed regions have limited access to meat and milk, so they would have problems taking enough vitamin B2 from daily food.

According to the Dietary Guidelines for Chinese Residents (2022), the Recommended Dietary Allowances for vitamin B2 are 1.4 mg/day for males and 1.2 mg/day for females. However, most people cannot have adequate vitamin B2 intake, and the vitamin B2 deficiency rate in China is as high as 88.1% in males and 85.4% in females (Ou et al., 2012). Vitamin B2 deficiency rate is affected by age, area, and dietary structure. Vitamin B2 deficiency is especially prevalent in several groups, including vegetarians, children, pregnant women, the elderly, and people from less developed regions (Li et al., 2020; Vudhivai et al., 1991; Tang et al, 2019).

Despite the importance of vitamin B2 for maintaining health and the prevalence of vitamin B2 deficiency, there appears to be a lack of awareness regarding this problem among the general population. The main vitamin B2 supplementation method is by medications, but people are not aware of vitamin B2 deficiency and therefore are not motivated to take medicine. Therefore, we decided to develop another method to mitigate vitamin B2 deficiency in China.

Survey of Vitamin B2 Supplement Awareness 

To investigate the public awareness of vitamin B2 supplements and deficiencies, we surveyed the public's attitudes towards vitamin B2, engineered yeast, and vitamin-B2-enriched food. We also intermewed a frequent vitamin B2 medicine user. To sum up, the results confirmed that most of the public was unaware of vitamin B2 deficiency. Based on the result, we also decided to hold some educational events to increase awareness. See https://2023.igem.wiki/hangzhou-biox/education for more details. Although vitamin pills can cure vitamin B2 deficiency rapidly, people are not aware of vitamin B2 deficiency and therefore are not motivated to take medicine. We found it inspiring that one-third of the respondents had tried genetically modified food before and the overall attitude towards it was positive.

Public Survey 

Respondents:

1169 people

Results and takeaways:

1) Only 39% of the respondents knew the symptoms of vitamin B2 deficiency (riboflavin deficiency, RD).

2) Only 19% of the respondents knew the Recommended Dietary Allowances (RDAs) for vitamin B2.

3) 39% of the respondents had never taken vitamin B2 supplements. Only 15% of the respondents took vitamin B2 supplements regularly, which was low considering the vitamin B2 deficiency rate in China is over 85%.

4) 77% of the respondents knew genetically modified food, and 36% had eaten it before, which indicated some degree of acceptance of GMOs.

Interview: 

Interviewee: Jufang Wang

62 years old

Frequent vitamin B2 medicine user

Results and takeaways:

1) Jufang used to suffer from cheilitis (inflammation of the lips) but did not know the cause.

2) Jufang went to see the doctor after reading an article about vitamin B and the doctor gave her vitamin B2 pills.

3) She failed to completely follow the instructions (three times/day) and often only took pills once or twice a day, but still recovered.

Research on Vitamin B2 Production 

Through literature research, we found that the mass production of vitamin B2 is mainly by microorganisms (Averianova et al., 2020). Natural and engineered bacteria (Bacillus subtilis, Escherichia coli, and lactic acid bacteria) and fungi (Eremothecium ashbyii and Ashbya gossypii) are used (Ledesma-Amaro et al., 2015; Hemberger et al., 2011; Liu et al., 2021; Hernández-Alcántara et al., 2020; Kapralek, 1962). The engineered stains often involve the modification of the pentose phosphate pathway, the purine nucleotide biosynthetic pathway, and the riboflavin synthesis pathway. The method gave us a clue about how to start our project.

In 2011, Capozzi et al. produced vitamin B2-enriched bread and pasta by adding riboflavin-overproducing strains of Lactobacillus plantarum during the bread-making process. Their product, the vitamin B2-enriched bread is just what we need to mitigate vitamin B2 deficiency in China. However, their method has flaws because adding extra lactic acid bacteria to the bread may change the production procedure and cause unexpected changes in taste. Innovated by them, we decided to make vitamin B2-enriched food in an easier way.

Bread and steamed buns are the main sources of carbohydrates for the Chinese people. They share similar ingredients: flour, water, and the baker's yeast (Saccharomyces cerevisiae). We planned to genetically modify the yeast to make vitamin-B2-enriched bread and steamed buns. The food fortification method for people to take vitamin B2 via daily food is perfect for solving vitamin B2 deficiency.

Technical Viability Check 

We found that S. cerevisiae has a natural riboflavin synthesis pathway (Gudipati et al., 2014). So, we only need to overexpress some enzymes without introducing exogenous genes. However, there are six genes directly controlling riboflavin synthesis, and there are more genes controlling the precursor of riboflavin and other competing pathways. We cannot manipulate all the related genes in this project. Therefore, we interviewed an expert in genetic engineering, and he suggested we target ADE4, RIB1, and RIB7. According to his suggestion, we also decided to use a prototrophic lab strain S. cerevisiae S288C.

Interview 

Interviewee: Dr. Yujun Wei

Doctor of Philosophy, Tsinghua University, School of Life Sciences

Occupation: CTO of Anlongbio.

Suggestions and takeaways:

1) We should use a prototrophic strain for our project.

2) Due to the complexity of riboflavin synthesis in S. cerevisiae, we should divide our project into several stages and only focus on a few genes in the first stage.

3) We could try to overexpress ADE4 from the purine nucleotide biosynthetic pathway, and RIB1 and RIB7 from the riboflavin synthesis pathway.

4) In the next stage, we could try to overexpress RIB2, RIB3, RIB5.

5) We should be careful about the vitamin B2 dose in food.

After determining the chassis and target genes, we turned to molecular cloning and vector design. Our primary design was to insert each gene downstream of a strong P_TEF1 promoter first, resulting in three vectors. Then, we planned to incorporate each expression cassette into one big vector, stacking two or three genes together. Finally, we transform the vectors into S. cerevisiae. We interviewed Prof. Zhu for advice. Since our original design would result in a vector larger than 11k bp, Prof. Zhu worried that it could cause vector construction and transformation difficulties. She recommended that we should do some research on polycistronic gene expression. We found that in eukaryotes, co-expression of multiple genes could be achieved by stacking multiple genes in one expression cassette. The 2A self-cleaving peptides could cleave a longer peptide into two shorter peptides. For the polycistronic gene expression system to work in our yeast, we planned to put the genes downstream of one P_TEF1 promoter and insert the 2A peptide sequences between them, with the stop codon of the prior genes deleted.

Interview 

Interviewee: Professor Xufen Zhu

Zhejiang University, Life Sciences Institute

Field of research: Microbiology and molecular biology

Suggestions and takeaways:

1) Our primary design was feasible, but not optimal.

2) The length of the inserted fragment should be taken into account since a very long one would lower the efficiency of vector construction, the rate of transformation, and the genetic stability.

3) We could improve our vector design by applying the polycistronic gene expression system.

Safety Considerations 

During communications with experts and potential customers, we have found that many are concerned about the risk of overdose on vitamin B2 if one eats too much vitamin B2-enriched food. After literature research, we concluded that an overdose of vitamin B2 is not harmful and very unlikely to happen. Firstly, the gut can only absorb a limited amount of riboflavin at one time (Institute of Medicine, 1998). Secondly, even if a high dose of vitamin B2 is absorbed, only small amounts of riboflavin are stored in the liver, heart, and kidneys. The excess will be easily excreted in the urine because vitamin B2 is water-soluble (Buehler, 2011). Therefore, a Tolerable Upper Intake Level for riboflavin has not been established and excessive intake of vitamin B2 does not harm the body (Dietary Guidelines for Chinese Residents, 2022).

Besides concerns about overdose, Prof. Qian pointed out the risk of toxic by-products produced by the engineered yeasts. Overexpression of ADE4 should lead to overexpression of IMP, which is the precursor of both GTP and ATP. Excess of GTP should lead to a higher amount of vitamin B2, and excess of ATP should facilitate cell growth as the energy carrier (Kowalski et al., 2008). RIB1 and RIB7 are only responsible for riboflavin synthesis, so overexpression of them would not lead to any unexpected product. Although we concluded that there should be no toxic by-products, we still need to do actual tests in the future and we plan to cooperate with the Center for Biosafety Research and Strategy.

Interview

Interviewee: Professor Zhao Qian

The Hong Kong Polytechnic University, Department of Applied Biology and Chemical Technology

Field of research: Proteomics

Suggestions and takeaways

1) Our project design dispelled some safety considerations about the engineered yeasts colonizing the human body because the yeasts will be killed during the process of baking or steaming.

2) We should carefully check if the overexpression of ADE4, RIB1, and RIB7 in S. cerevisiae leads to any toxic by-products other than riboflavin.

Regulations and National Approval Process

As food made with genetically modified organisms, it was meant to be a special product that acquired unusual restrictions. The first question was whether our engineered yeast could meet the regulatory requirements. Then, the vitamin-B2-enriched bread and buns must also be approved. We did literature research to find the regulations and evaluation processes for engineered organisms and new food raw materials. To conclude, the vitamin-B2-enriched food made with our yeast has to go through two rounds of assessment. The yeast's safety as a genetically modified organism will be evaluated first. After approval, we can move to the next step, which is to evaluate the safety of our yeast as a food material. After that, we can use our yeast to make bread and buns and sell them in supermarkets. Although complicated, the yeast and food can be approved if we comply with all the regulations

Literature research 

1) Our yeast must pass three stages of trials: intermediate test, environmental release test, and production test (the Measures for the Administration of the Safety Evaluation of Agricultural Genetically Modified Organisms (2016 Revision, Article 13).

2) The State Commission for the Safety of Agricultural Genetically Modified Organisms and the Office for the Safety Management of Agricultural Genetically Modified Organisms are responsible for the general safety evaluation of our yeast, and we must submit the Application for the Safety Evaluation.

3) The Ministry of Agriculture and Rural Affairs is responsible for issuing the Agricultural Genetically Modified Organisms Safety Certificate.

4) The Chinese National Center for Food Safety Risk Assessment is responsible for the safety evaluation of our yeast as a food raw material, and we must submit the Application for the Evaluation of New Food Raw Materials (Administrative Measures for the Safety Review of New Food Raw Materials (2017 Amendment), Article 6).

5) The Chinese National Center for Food Safety Risk Assessment will publish the  Consultation Paper on our yeast (Administrative Measures for the Safety Review of New Food Raw Materials (2017 Amendment), Article 6).)

6) The National Health Commission is responsible for issuing the Approved List of New Food Raw Materials.

Product Design 

Finally, since we planned to produce bread and buns with our yeast in the future, current bakeries could be our peers and cooperators. We would like to know more about the technical details of bread-making and information about the bakery industry to evaluate our project's future. So, we interviewed a local bakery and expert in food nutrition, asking for their ideas about our plan. The conversations changed our minds about our final product. We figured that we needed to create distinct products for various target groups. They believed that providing yeast or baking mix was more feasible than providing food. They suggested we consider the economic resources of people in rural areas and provide them with relatively inexpensive yeast powder products. We also plan to sell yeast to food producers. Prof. Huang mentioned baking mixes as a new trend in the food industry. We plan to design a variety of baking mixes for different groups of customers, such as low-sugar mixes for dieters, high-calcium mixes for the elderly and children, etc. All the suggestions helped us determine our product design. We have designed a business plan as well. Please refer to https://2023.igem.wiki/hangzhou-biox/entrepreneurship for further details.

Interview 

Interviewee: Yalin Liu

Occupation: Store manager of Koi Fish Bread, Haining, Zhejiang, China

Suggestions and takeaways:

1) Unlike the instant dry yeast sold in supermarkets to general customers, the more efficient fresh yeast was used in bakeries.

2) Yalin suggested that we could become yeast suppliers instead of bakeries since running a bakery was difficult because it was very customer-oriented.

3) Yalin would love to make bread with our engineered yeast if it got approved.

Interview 

Interviewee: Professor Huang

Nanjing Tech University, College of Food Science and Light Industry

Field of research: Food safety and nutrition

Suggestions and takeaways:

1) We can cooperate with the local government of rural areas, and sell our product as yeast powder at a very low price. This can work as a public welfare program.

2) Baking mixes (pre-mixed flour, yeast, sugar, etc) are also popular products and have a big market.

3) We can produce a variety of baking mixes for different groups of customers. We can produce low-sugar mix for dieters, high-calcium mix for the elderly and children, etc.

3) We can add natural pigments to the baking mix products designed for young children to increase children's interest and appetite.

References