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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. 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.

Contents 1 The Problem with Rice 2 The Plant GM Approach to Fortification 3 The iRICE System 4 Vitamin B12-Binding Protein 5 Thiamine-Binding Protein 6 Lysine-Rich Protein 7 Proof of Concept 8 References

The Problem with Rice

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 nearly 205 calories.

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 in the production of white rice. 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?

The Plant GM Approach to Fortification

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, an essential vitamin for retinal formation and light transduction in the eye. The concept of genetically modifying is conceptually simple: simply add the genes for beta-carotene production into the genome for rice and grow the new vitamin A enriched rice strain.

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 unfortified white rice. 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 the iRICE.

The iRICE System

The idea behind this project was to create a protein that can bind nutrients to rice and can be purified much like a pharmaceutical before being added to rice. The vitamin binding protein will then be coated onto rice grains after the milling process. The fusion 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. This is where the customization comes in. The combinations of binding domains you can use is diverse. In our case we have choosen thiamine and B12 (cobalamin) binding domains, but one could use any number of vitamins, amino acid-rich proteins, or oral vaccine, such as cholera or hepatitis B.

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 plants exists should popular opinion change in the future. Rather than create supplemental proteins that must be added in the milling process the vitamin binding proteins would be expressed by the plants themselves.

Vitamin B12-Binding Protein

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.

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.

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.

Thiamine-Binding Protein

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.

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.

Where do Americans get most of their thiamine? Surprisingly, the answer is cereal. Vitamin fortified cereal is distributed by major brands such a General Mills, Kashi, and Kellogg’s, who all fortify their cereal with thiamine. Over 50% of the United States relies solely on breakfast cereal to get thiamine in their diet. 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 in the mornings instead. 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 our 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 the rice and help provide this essential vitamin.

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. 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. 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, enriching lysine intake in the diets of people worldwide.

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.

For more on our RFP-SBP proof of concept results as well as quantitative binding data for one of our starch-binding nutrient constructs, please check out our results.

References

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.

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.

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.

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.

Hedhammar, M. and S. Hober. 2007. Z(basic) –a novel purification tag for efficient protein recovery, J. Chromatogr. A. 1161: 22-28.

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. 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.

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.

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.