Team:Nevada/Project/

iRICE: A Novel Approach to Biofortification of Rice
Even though white rice is a major source of calories for over half the world’s population, it is a poor source of nutrients. This is due to the loss of nutrients during milling and polishing. Fortification of white rice is a common practice in the US and typically involves a vitamin dusting technique as the primary method for supplementation [1] . Such approaches have had limited success in most rice dependent cultures because most people wash their rice prior to cooking, and many vitamins are leeched away during the washing process [1] . To address this problem, we have engineered proteins that will adhere nutrients to rice grains and prevent losses. These adhesion proteins contain a starch-binding domain that is fused to specific nutrient-binding domains. Because rice is composed mainly of starch, the starch-binding domain prevents nutrient leeching during washing. Supplementing rice with these adhesion proteins will provide an alternative approach to fortifying rice. Proteins with a starch-binding domain connected to a Vitamin B12-binding domain, a thiamine-binding domain, a lysine-rich protein, and a Red Fluorescent Protein have been the major focus of this project.

The Problem with Rice

Providing adequate nutrition for healthy living is a global issue. Half the world's population (over 3 billion people) depends on rice to survive. In Asia, much of the population consumes rice in every meal, and rice accounts for greater than 70% of human caloric intake in countries such as Cambodia, Bangladesh, and Myanmar. White rice consists of 85% carbohydrates, 7% fat, and 8% protein. One cup of this grain can yield over 200 calories [1] .

From rice in fields to rice on your plate, there are numerous procedures that rice must undergo before it is ready to be eaten. The first process is called milling, which removes the husk and bran layers of rice. The husk is the shell, a hard non-edible layer that protects the edible bran and endosperm. The bran layer is where most of the nutrition is located and it is removed in the production of white rice [2]. Leaving the bran layer intact produces brown rice, but many people from rice dependent cultures do not like the taste of brown rice or they consider it the rice of the poor. Instead, they prefer white rice, which contains virtually no nutritional value. So how do we fix this?

The Plant GM Approach to Fortification

A modern approach to add nutrition to food is through genetically modifying the crop itself. An example of this is Golden Rice, a rice grain engineered to produce beta-carotene (pro-vitamin A), an essential vitamin for retinal formation and light transduction in the eye. The idea of genetically modifying is conceptually simple: add the genes for beta-carotene production into the genome for rice and grow the enriched rice strain to combat vitamin A deficiency[3].

However, there are some disadvantages to genetically modified plants, such as the possibility of horizontal gene transfer and the limiting nature of genetically modifying one cultivar at a time. The biggest disadvantage of transgenic plants is that most people and cultures don’t accept these transgenic species. Africa won’t allow transgenic grains into their continent, India refuses to eat Golden Rice, and other countries in Asia simply prefer the taste of unfortified white rice. There is a widespread negative stigma against genetically modified plants, which makes products such as Golden Rice very difficult to introduce to the public. This is why there is a need for a new approach to fortified rice that does not alter the natural genetic code of rice itself and can still provide just as much nutrition as a genetically modified strain. This is why we developed iRICE.

The iRICE System

The idea behind this project was to create an adhesion protein that can be added to rice to help bind nutrients to the grain. Although this protein is genetically engineered, it is fully produced in a lab setting using microorganisms and then purified, much like a pharmaceutical, and then will be sprayed onto milled rice, rather than being engineered into the rice itself. The iRICE will be coated onto rice after the final stage of the milling process just prior to packaging. The iRICE protein consists of a starch binding domain that will adhere to (in this case) the starch in rice, and one of any number of vitamin binding or enrichment proteins [4]. This is where the customization comes in. The combinations of binding domains you can use is diverse. In our case we have chosen thiamine and B12 binding domains, but one could use any number of vitamins, amino acid-rich proteins (lysine-rich in our project), or oral vaccine, such as cholera or hepatitis B [5].

Given the current opinion towards genetically modified organisms (GMOs) we have designed our constructs to be used as a supplement, rather than expressing the protein in rice plants. However, the potential for GMO plants is still an option if popular opinion changes in the future [6]. Rather than create supplemental proteins that must be added after the milling process, iRICE would be expressed by the plants themselves.

Vitamin B12-Binding Protein

Vitamin B12, Cyanocobalamin, is a vitamin involved in many neurological processes. In addition, it is crucial in the development and maintenance of key parts within the nervous system [7]. The main source of bioavailable B12 is found in meat and dairy products. While it is found in plant sources, it is not biologically usable by humans or other mammals. This poses a problem for vegans or people whose main calorie source comes from non-animal products. This includes populations dependent on rice or rice products [8].

While deficiencies in vitamin B12 are not well documented, it is slowly becoming a more relevant issue. Often times, B12 deficiencies mimic ailments that are closely related to Alzheimer’s and dementia, or more generally, symptoms related to fatigue. This inhibits the ability to target and treat B12 deficiency symptoms. It is proposed that the current recommendations, 200-350 pg/ml, in USA are on the low level and that 40% of people between 26 and 80 are at or below this level. Within this range, symptoms begin to manifest, and depending on the longevity of this sustained deficiency, ailments will become increasingly severe. If the deficiency is severe enough permanent brain damage can occur. The issue arises in a more relevant manner when a population dependent on rice and its bi-products is considered as rice contains no detectable amounts of vitamin B12 [6].

There are no current methods used to fortify rice with B12 which is why projects like this one are innovative. The new protein created in this project utilizes the starch binding capabilities of the CBM 21 gene, coding for a starch binding protein, as well as the BtuF gene coding for B12 binding protein. Together these proteins will effectively bind starch and supplement rice, or any number of starch products, with vitamin B12.

Thiamine-Binding Protein

Thiamine, or vitamin B1, is an essential vitamin for healthy living and proper development of the nervous and cardiovascular system [9]. Thiamine is a necessary co-factor in our body’s metabolic pathway for breaking down carbohydrates into energy [10]. Thiamine deficiency presents itself as baribari disease and Wernicke-Korsakoff syndrome. Beriberi disease has many forms but is most notably characterized by loss of muscle function, pulmonary edema, and hypotension which may lead to death. A diet consisting of healthy thiamine levels may help in prevention of cataracts, Alzheimer’s disease, and kidney damage [6].

Surprisingly, Americans get most of their thiamine from cereal. Vitamin fortified cereal is distributed by major brands such a General Mills, Kashi, and Kellogg’s, who all fortify their cereal with thiamine. But many people in other countries don't eat cereal for breakfast. In fact, in areas where rice is a staple grain, they often eat leftover rice for their morning meal. There is some thiamine in the outer husk of rice, but this is lost during washing because thiamine is water soluble. This is why thiamine is crucial for the iRICE project. By creating a new gene that fuses a thiamine-binding protein and a starch-binding protein together, we hope to increase the retention rate of thiamine in rice and help provide this essential vitamin to millions of people.

Lysine-Rich Protein

Lysine is an essential amino acid required for growth and bone development, tissue repair, and producing antibodies, hormones, enzymes, and collagen. Lysine also reduces the symptoms of herpes infections, stress-induced anxiety, and decreases harmful LDL cholesterol levels by aiding in the production of carnitine [11]. Lysine is obtained through meat products and through beans and legumes for people on a strict low-meat diet. This essential amino acid is limiting in the diets of vegans and people who are on a wheat-based diet.

A diet deficient in lysine could lead to osteoporosis, fatigue, anemia, hair loss, reproductive disorders, and heart problems for people who cannot obtain the daily recommended intake of lysine through their diet [12]. The recommended daily allowance for lysine is 12 mg per kg of body weight. In rice, there is about 145 mg lysine per serving. Therefore, the average adult male would have to consume nearly 6 servings of rice a day to meet minimum lysine levels. With rice as the main staple food source for a majority of the populations in the world, it is vital to fortify rice with lysine to prevent lysine deficiency worldwide.

Therefore in this project, the starch binding capabilities of the starch-binding protein gene, CBM 21, and the lysine-rich protein gene, ABY71635.1, will effectively bind to the starch in rice to supplement rice [13]. Unlike the vitamin-binding proteins, this fusion would directly supplement rice with Lysine. This protein is still currently under construction.

Proof of Concept

References

1.  ; Available from: http://www.ift.org/Knowledge-Center/Read-IFT-Publications/Science-Reports/Contract-Reports/Rice-Fortification-in-Developing-Countries.aspx?page=viewall.
2. Ellis, J.R., C.P. Villareal, and B.O. Juliano, Protein content, distribution and retention during milling of brown rice. Plant Foods for Human Nutrition (Formerly Qualitas Plantarum), 1986. 36(1): p. 17-26.
3. Mayer, J.E., Delivering golden rice to developing countries. J AOAC Int, 2007. 90(5): p. 1445-9.
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5. Chaiken, M.S., Beriberi, White Rice, and Vitamin B: A Disease, a Cause and a Cure. Nutritional Anthropology, 2001. 24(1): p. 12-13.
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8. G. F. M. Ball, in: Bioavailability and Analysis of Vitamins in Foods, Chapman and Hall, London, 1998, p. 498
9. Iwashima, A., A. Matsuura, and Y. Nose, Thiamine-binding protein of Escherichia coli. J Bacteriol, 1971. 108(3): p. 1419-21.
10. Hirota, Y., K. Thorp, and W.H. Abelmann, Protective effect of coexistent thiamine deficiency upon the experimental cardiomyopathy associated with acute magnesium deficiency in the Syrian golden hamster. Recent Adv Stud Cardiac Struct Metab, 1975. 10: p. 695-706.
11. Klemesrud, M.J., T.J. Klopfenstein, and A.J. Lewis, Metabolize methionine and lysine requirements of growing cattle. J Anim Sci, 2000. 78(1): p. 199-206.
12. Simell, O., et al., Lysinuric protein intolerance. Am J Med, 1975. 59(2): p. 229-40.
13. Machovic, M., et al., A new clan of CBM families based on bioinformatics of starch-binding domains from families CBM20 and CBM21. FEBS J, 2005. 272(21): p. 5497-513.