Team:Nevada/Project/

From 2012.igem.org

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<b>iRICE: A Novel, Non-Plant GM Approach to Biofortification of Rice</b>
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<b>iRICE: A Novel Approach to Biofortification of Rice</b>
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Even though white rice is a major source of calories for over half the world’s population, it is a poor source of nutrients. While rice can be fortified using vitamin powders, such approaches have had limited success because many vitamins are leeched away during the washing process prior to cooking. To address this problem, we have engineered proteins that will adhere nutrients to rice grains and prevent losses. These 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. Upon cooking, the nutrient-binding domain denatures and releases the nutrients into the cooked rice. Supplementing rice with these fusion proteins will provide a non-plant GM 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 RFP have been created.  
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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.</html>
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==The Problem with Rice==
==The Problem with Rice==
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Providing adequate nutrition for healthy living is an issue in developing countries. About half the world's population (3 billion people) depend on rice to survive. In Asia, much of the population consumes rice in every meal. 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 5 grams of protein and nearly 205 calories.
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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] .
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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?
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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. Milling is the removal of the husk and bran layer 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 most of the times removed due to taste purposes. Many people from Asian countries do not like the taste of brown rice or they consider it the rice of the poor. Many people prefer white rice which contains virtually no nutritional value. So how do we fix this?
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==The Plant GM Approach to Fortification==
==The Plant GM Approach to Fortification==
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The most modern approach to providing nutrition through food now is through genetically modifying the rice itself. An example of this is Golden Rice, a rice grain fortified with beta-carotene which provides vitamin A when cooked. The concept of genetically modifying is conceptually simple, simply add the genes for beta-carotene production into the genome for rice and grow your vitamin A enriched rice.  
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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].  
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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.
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The biggest problem with genetically modified 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 colored rice, and other countries in Asia simply prefer the taste of white rice that lacks any nutrition. This is why there is a need for a new grain of rice that does not alter the natural genetic code of rice and can still provide just as much nutrition as genetically modified. This is why we developed the iRICE.
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==The iRICE System==
==The iRICE System==
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The starch binding gene used in this project, CBM 21, is derived from the species, R. oryzae, which is a form of fungus that thrives on dead organic matter, including starch containing species. It is part of an operon that codes for a two-domain enzyme. One domain has the catalytic function of breaking down starch while the other domain binds to the starch molecule in order to provide optimal conditions for the catalytic portion. This project takes advantage of the starch binding domain encoded by this gene and it is utilized for its ability to bind rice starch efficiently.  
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<html><center>
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<img src="https://static.igem.org/mediawiki/2012/3/36/IRICEfigv2.png"></img></center></html>
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The idea behind this project was to create a complex that is not only effective in nutrient enhancement but adaptable to the specific needs of various populations. The construct consists of a starch binding protein that will adhere to (in this case) the starch in rice, and one of any number of binding proteins. This is where the customization comes in. The combinations or binding proteins you can use is diverse. In our case we have used Thiamine and Cobalamin binding proteins that will adhere available vitamins from the environment, but one could use any number of vitamin, amino acid, or pharmaceutical (speculatively) binding proteins.
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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].
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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.
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Given the current opinion towards GMOs we have designed our constructs to be used as an additive that could be used in a coating or dusting process. However, the potential for GMO exists should popular opinion change in the future. Rather than create supplimental proteins that must be added in the milling process the vitamin binding proteins would be expressed by the plants themselves.
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==Vitamin B12-Binding Protein==
==Vitamin B12-Binding Protein==
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Vitamin B12, Cyanocobalamin, is a vitamin involved in many neurological processes. The main source of dietary 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 problems for those who follow vegan diets or whose main calorie source comes from non-animal products. This includes populations dependent on rice or rice products.
 
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While deficiencies in vitamin B12 are not well documented, it is slowly becoming a more crucial problem. Often times, B12 deficiencies mimic ailments that are closely related to Alzheimer’s and dementia, or more generally, symptoms related to fatigue. It is proposed that the current recommendations in USA are on the low level, and that 40% of people between 26 and 80 are at or below this level. At these levels, symptoms begin to manifest, and, depending on the longevity of this sustained deficiency, permanent brain damage may occur. This is alarming when considering populations dependent on rice as there is no B12 present.  
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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].
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<Br><Br>
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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].
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<Br><Br>
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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.
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There are no current methods used to fortify rice with B12 which is why this project is innovative. It 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.  
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<html><center><img src="https://static.igem.org/mediawiki/2012/b/bc/SBP-B12BP.jpg"></center></html>
==Thiamine-Binding Protein==
==Thiamine-Binding Protein==
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Thiamine or vitamin B1 is an essential vitamin for proper growth and development. Thiamine is a necessary cofactor for our body so that it can break down carbohydrates to glucose properly and make ATP which provides us with energy.  
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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].
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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.
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Some of the harmful effects of thiamine deficiency are diarrhea, stomach problems, and even difficulty walking. But, a big risk associated with thiamine deficiency is Beriberi. Beriberi is a disease that causes loss of muscle function, loss of sensation in the hands and feet, and even mental confusion. Thiamine also helps in prevention of cataracts, cervical cancer, and kidney damage.
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Why is thiamine a concern if only the alcoholics are the only ones with a thiamine deficiency? A better question to ask is where do we get most of our thiamine...? Cereal. Vitamin fortified cereal, this includes majors brands such a General Mills, Kashi, and Kellogg’s all fortify their cereal with thiamine. Over 50% of our nation relies solely on breakfast cereal to get thiamine in their diet. What about other countries that don’t eat break cereals? This is why thiamine is crucial for our iRICE project. An alternative approach to providing one of the eight essential vitamins is a more acceptable form of proper nutrition compared to take vitamin pills.
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==Lysine-Rich Protein==
 
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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. 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.
 
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<html><center><img src="https://static.igem.org/mediawiki/2012/6/62/SBP-TBP.jpg"></center></html>
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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. 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.
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==Lysine-Rich Protein==
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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, enriching lysine intake in the diets of people worldwide.  
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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.
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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.  
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==Proof of Concept==
 
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Red Fluorescent Protein is a protein that glows red when exposed to visible light. RFP and other fluorescent proteins are used frequently in biological experiments as marker proteins.
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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.
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RFP was combined with Starch binding protein successfully. The construct was used to show that the starch binding protein can bind to rice. After binding, we also used an amylase column to show binding.  
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<html><center><img src="https://static.igem.org/mediawiki/2012/e/e4/SBP-LRP.jpg"></center></html>
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==Proof of Concept==
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Each of the proteins was expressed successfully with a starch binding domain and their nutrient binding components shown to bind their nutrient. The red fluorescent protein shows the functionality of the starch binding protein. With both domains shown to be working, our project comes to a successful close.
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<html><center><img src="https://static.igem.org/mediawiki/2012/a/ae/RFP-iPhone-pic1.jpg"></img></center>
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==References==
 
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Berntsson, R., S. Smits, L. Schmitt, D-J Slotboom, and B. Poolman. 2010. A structural classification of substrate-binding proteins, FEBS Letters 584: 2606-2617.
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Red Fluorescent Protein is a protein that appears red to the naked eye and glows under exposure to UV light. RFP and other fluorescent proteins are used frequently in biological experiments as marker proteins. RFP was combined with starch binding protein in a similar method as the SBP-nutrient protein fusions, with RFP on the C-Terminus of the protein and SBP on the N-Terminus. This construct was used to visually demonstrate the ability of the fusion protein to bind to rice via the starch-binding domain. The picture above is an example of the kind of results we are looking for.
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For more on our RFP-SBP proof of concept results and of our starch-binding nutrient constructs, please check out our <a href="https://2012.igem.org/Team:Nevada/Results/">results</a>.
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Chou, W.I., T.W. Pai, S.H. Liu, B.K. Hsiung, and M.D. Chang. 2006. The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites &nbsp &nbsp &nbsp &nbsp &nbsp  playing distinct roles in ligand binding, Biochem. J. 396: 469-477.
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==References==
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Cadieux, N., C. Bradbeer, E. Reeger-Schneider, W. Koster, A.K. Mohanty,  M.C. Wiener, and R.J. Kadner1. 2002. Identification of the Periplasmic Cobalamin-Binding Protein BtuF of Escherichia coli. American Society for Microbiology 184: 243-247.
 
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1.   ; Available from: http://www.ift.org/Knowledge-Center/Read-IFT-Publications/Science-Reports/Contract-Reports/Rice-Fortification-in-Developing-Countries.aspx?page=viewall.
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Gottlieb, C., K-S Lau, L.R. Wasserman and V. Herbert. 1965. Rapid Charcoal assay for intrinsic factor (IF) gastric juice unsaturated B12 binding capacity, antibody to IF, and serum unsaturated B12 binding capacity. Blood J. Hematol. 25: 875-884.
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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.
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Hedhammar, M. and S. Hober. 2007. Z(basic) –a novel purification tag for efficient protein recovery, J. Chromatogr. A. 1161: 22-28.
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3. Mayer, J.E., Delivering golden rice to developing countries. J AOAC Int, 2007. 90(5): p. 1445-9.
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4. Lin, S., et al., CBM21 starch-binding domain: A new purification tag for recombinant protein engineering. Prtoein Expression and Purification, 2009. 65: p. 261-66.
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Li, H-B, F. Chem, and Y. Jiang. 2000. Determination of vitamin B12 in multivitamin tablets and fermentation medium by high-performance liquid chromatography with fluorescence detection. J. Chromatography A. 891: 243-247.
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Lin, S-C., I-P Lin, W-I Chou, C-A Hsieh, S-H Liu, R-Y Huang, C-C Sheu, and M. Dah-Tsyr Chang. 2009. CMB21 starch-binding domain: A new purification tag for recombinant protein engineering, Protein Expression and Purification 65: 261-266.
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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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6. Tenbult, P., et al., Acceptance of genetically modified foods: the relation between technology and evaluation. Appetite, 2008. 51(1): p. 129-36.
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Locher, K., A. Lee, and D. Rees. 2002. The E. coli BtuCD structure: A framework for ABC transporter architecture and mechanism. Science 296: 1091-1098.
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<br>
-
 
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7. Cadieux, N., et al., Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli. J Bacteriol, 2002. 184(3): p. 706-17.
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<br>
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Nathan C Shaner et al, Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, Nature Biotechnology 22 (2004), 1567 - 1572.
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8. G. F. M. Ball, in: Bioavailability and Analysis of Vitamins in Foods, Chapman and Hall, London, 1998, p. 498
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9. Iwashima, A., A. Matsuura, and Y. Nose, Thiamine-binding protein of Escherichia coli. J Bacteriol, 1971. 108(3): p. 1419-21.
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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.
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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.
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12. Simell, O., et al., Lysinuric protein intolerance. Am J Med, 1975. 59(2): p. 229-40.
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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.

Latest revision as of 23:41, 26 October 2012



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.

Contents

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

Red Fluorescent Protein is a protein that appears red to the naked eye and glows under exposure to UV light. RFP and other fluorescent proteins are used frequently in biological experiments as marker proteins. RFP was combined with starch binding protein in a similar method as the SBP-nutrient protein fusions, with RFP on the C-Terminus of the protein and SBP on the N-Terminus. This construct was used to visually demonstrate the ability of the fusion protein to bind to rice via the starch-binding domain. The picture above is an example of the kind of results we are looking for.

For more on our RFP-SBP proof of concept results and of our starch-binding nutrient constructs, please check out our results.

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.
4. Lin, S., et al., CBM21 starch-binding domain: A new purification tag for recombinant protein engineering. Prtoein Expression and Purification, 2009. 65: p. 261-66.
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.
6. Tenbult, P., et al., Acceptance of genetically modified foods: the relation between technology and evaluation. Appetite, 2008. 51(1): p. 129-36.
7. Cadieux, N., et al., Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli. J Bacteriol, 2002. 184(3): p. 706-17.
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.