Vitamin B2, also known as riboflavin, is one of the most important vitamins. Vitamin B2 deficiency can lead to inflammation and
organic disorders of the mouth, lips, skin, and genitalia. Despite the importance of vitamin B2 for maintaining optimal health,
there appears to be a lack of awareness regarding vitamin B2 deficiency among the general population. In China, the vitamin B2
deficiency rate is as high as 88.1% in males and 85.4% in females, and most of them are not aware of it (Ou et al., 2012). To
deal with the prevalence of vitamin B2 deficiency, we decided to genetically modify the baker's yeast to make vitamin-B2-rich
bread and steamed buns. Our project aims to achieve a high yield of vitamin B2 in Saccharomyces cerevisiae by overexpressing GTP synthetase (ADE4) and vitamin B2 synthetase (RIB1 and RIB7). Food producers and bakeries can produce
bread and steamed buns by using our yeast at no extra cost, and supply vitamin-B2-enriched food for the general public.
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. Chronic riboflavin deficiency in children results in slow growth and iron deficiency anemia. Notably, severe
deficiency of vitamin B2 is often accompanied by deficiencies of other B vitamins (Xiang et al., 2004).
Figure 1. Structure of vitamin B2.
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. In China, the
vitamin B2 deficiency rate is as high as 88.1% in males and 85.4% in females (Ou et al., 2012).
On the other hand, an overdose of vitamin B2 is not practical because the gut can only absorb a limited amount of riboflavin at one time, and excess is quickly excreted in the urine. Therefore, a
Tolerable Upper Intake Level for riboflavin has not been established and excessive intake of vitamin B2 does not harm the
body.
Despite the importance of vitamin B2 for maintaining health, there appears to be a lack of awareness regarding vitamin B2
deficiency among the general population. A population-based study by McNulty et al. (2017) reported that only a small
number of participants took riboflavin supplements, although their dietary intake was below the recommended daily intake. What
makes the situation even worse is that many doctors know little about vitamin B2 deficiency, and cannot give appropriate
suggestions.
|
Methods of vitamin B2 intake |
Disadvantages |
|
By consuming meat, eggs, and milk. |
It is restricted by economic conditions. Poverty-stricken areas do not have enough money to buy those foods. |
|
Some people are lactose intolerant. They cannot gain vitamin B2 from dairy products. |
|
|
By taking medications to supplement vitamin B2 |
People are not aware of vitamin B2 deficiency and therefore are not motivated to take medicine. |
|
Although not much, vitamin B2 pills cost money |
Table 1. Methods of vitamin B2 intake.
As discussed above, the prevalence of vitamin B2 deficiency cannot be mitigated by medicine due to the lack of awareness.
Therefore, we must develop a method for people to take vitamin B2 via daily food. Food fortification is a suitable option. Like
vitamins D and A being added to milk sold in the United States, we want to add vitamin B2 to daily food.
Bread and steamed buns are the main sources of carbohydrates for the Chinese people. They share similar ingredients: flour,
yeast, and water. Yeast is our target. We plan to increase the vitamin B2 production in yeasts to make the vitamin-B2-enriched food.
Saccharomyces cerevisiae, the baker's yeast, is one of the most commonly used microorganisms in the world. It has been used in bakery and wine
production for thousands of years and has been proven safe for humans.
Since we want the yeast to be able to reproduce in the kitchen environment and rise dough, we chose the S. cerevisiae S288C. Unlike many auxotrophic laboratory strains of S. cerevisiae, S288C is a prototrophic strain. S288C is also fully sequenced and well-studied, so we should be able to get predictable
results after molecular manipulations.
Another advantage of using S. cerevisiae to produce vitamin B2 is that it has a natural riboflavin synthesis pathway (Gudipati et al., 2014). So, we only need to
overexpress the riboflavin synthases without introducing exogenous genes.
To increase the amount of vitamin B2 production in S. cerevisiae S288C, we first looked at the riboflavin synthesis pathway. There are six main riboflavin synthases discovered, named
RIB1, RIB7, RIB2, RIB3, RIB4, and RIB5 (Gudipati et al., 2014). Although no research has shown that the overexpression of these
genes could increase the riboflavin production of S. cerevisiae, researchers have found that overexpressing the RIB genes (homologs genes) in Ashbya gossypii and Pichia pastoris led to a higher amount of riboflavin production (Ledesma‐Amaro et al., 2015; Marx et al., 2008). This year, we
overexpressed RIB1 (GTP cyclohydrolase II) and RIB7 (diaminohydroxyphoshoribosylaminopyrimidine deaminase) in S. cerevisiae.
Figure 2. The GTP synthesis pathway and the riboflavin synthesis pathway of S. cerevisiae (Kowalski et al., 2008; Gudipati et al., 2014)
GTP is the precursor of vitamin B2 (Kowalski et al., 2008). So, in theory, increasing the amount of GTP could also increase the
amount of vitamin B2 in S. cerevisiae. We chose to overexpress ADE4, which encodes the phosphoribosylpyrophosphate amidotransferase.
TEF1 promoter is from the yeast Yarrowia lipolytica that controls the expression of the translation elongation factor-1 alpha, a protein that is one of the most expressed in
most cells (Müller et al., 1998). Ptef1 has been proven one of the strongest promoters of S. cerevisiae, so we used it for overexpression (Partow et al., 2010).
The results showed that the overexpression of any of the ADE4, RIB1, and RIB7 helped to increase vitamin B2 production. Stacking all three genes gave the highest vitamin B2 yield.
Vitamin B2 deficiency, one of the biggest threats to health in China, can be tackled by vitamin-B2-enriched food produced by
our engineered yeast. By using our modified yeast to produce bread and steamed buns, bakeries and food producers can supply vitamin-B2-enriched food
for the general public without changing any steps or paying for anything else.
This year, due to the time limit, we only overexpressed three genes. Research has shown that the more RIB genes get
overexpressed, the more vitamin B2 got produced in yeasts (Ledesma‐Amaro et al., 2015; Marx et al, 2008). So our project
can be further improved in the further by overexpressing all the six known riboflavin synthases in S. cerevisiae.
Gudipati, V.; Koch, K.; Lienhart, W.-D.; Macheroux, P. The Flavoproteome of the Yeast Saccharomyces Cerevisiae. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2014, 1844 (3), 535-544. DOI:10.1016/j.bbapap.2013.12.015.
Kowalski, D.; Pendyala, L.; Daignan-Fornier, B.; Howell, S. B.; Huang, R.-Y. Dysregulation of Purine Nucleotide Biosynthesis
Pathways Modulates Cisplatin Cytotoxicity in Saccharomyces Cerevisiae. Molecular Pharmacology 2008, 74 (4), 1092-1100. DOI:10.1124/mol.108.048256.
Ledesma-Amaro, R.; Serrano-Amatriain, C.; Jiménez, A.; Revuelta, J. L. Metabolic Engineering of Riboflavin Production in
Ashbya Gossypii through Pathway Optimization. Microbial Cell Factories 2015, 14 (1). DOI:10.1186/s12934-015-0354-x.
Marx, H.; Mattanovich, D.; Sauer, M. Overexpression of the Riboflavin Biosynthetic Pathway in Pichia Pastoris. Microbial Cell Factories 2008, 7 (1). DOI:10.1186/1475-2859-7-23.
McNulty H, Strain JJ, Hughes CF, Ward M. Riboflavin, MTHFR genotype and blood pressure: A personalized approach to prevention
and treatment of hypertension. Mol Aspects Med. 2017;53:2-9. doi:10.1016/j.mam.2016.10.002
Müller, S.; Sandal, T.; Kamp-Hansen, P.; Dalbøge, H. Comparison of Expression Systems in the Yeastssaccharomyces
Cerevisiae, Hansenula Polymorpha, Klyveromyces Lactis, Schizosaccharomyces Pombe Andyarrowia Lipolytica. Cloning of Two Novel
Promoters Fromyarrowia Lipolytica. Yeast 1998, 14 (14), 1267-1283.
Ou, F.; Ling, L.; He, Y.; Yang, X.; Xu G.; Ma, G. Assessment of Nutrient Adequacy of Adult. Acta Nutrimenta Sinica 2012, 34(1):15-19
Parapouli, M.; Vasileiadi, A.; Afendra, A.-S.; Hatziloukas, E. Saccharomyces cerevisiae
and Its Industrial Applications. AIMS Microbiology 2020, 6 (1), 1-32. DOI:10.3934/microbiol.2020001.
Partow, S.; Siewers, V.; Bjørn, S.; Nielsen, J.; Maury, J. Characterization of Different Promoters for Designing a New
Expression Vector in Saccharomyces Cerevisiae. Yeast 2010, 27 (11), 955-964. DOI:10.1002/yea.1806.
Xiang Zhaobao, Dai Chuanyun, Zhu Liqing, Physiological and biochemical characteristics of riboflavin and its functions, Food
Research and Development, 2004 (06)
