Genetic Engineering

Genetic Engineering of Organism

Genetically Engineering Folate Biofortification in Tomato Claudia Lerma-Ortiz

Tetrahydrofolate (THF) and its derivatives (folates) are indispensable for the health of humans and other organisms. They participate as co factors in one carbon transfer reactions in the synthesis of glycine, serine, methionine, purines, and thymidylate1,2. Humans are unable to synthesize folates. A deficiency of this vitamin can cause a range of serious diseases, including some birth defects (such as spina bifida), megaloblastic anemia, cardiovascular conditions, and some cancers3.

Although folate deficiency is still a serious problem all over the world, it has the highest impact in developing countries, causing 200,000 severe birth defects every year. To help prevent folate deficiency, some western countries, including the United States, have made the fortification of grain products with synthetic folic acid mandatory. Fortification helps people get the recommended allowance—400 μg/day for adults and 600 μg/day for pregnant women—in their regular diet. Unfortunately, in developing countries this kind of fortification presents a series of challenge distribution inequities, recurrent costs, and lack of a food industry. Furthermore, fortification may itself cause some health problems. When a large amount of this vitamin is ingested, unreduced folic acid can enter into the systemic circulation4. Therefore, to achieve a greater margin of safety and lower costs, the consumption of common foods with a high content of natural THF would be a better solution.

Another consideration is that most folate in people’s diets comes from plant foods. However, people regularly consume tubers, cereals, and fruits and not green leafy vegetables, which have the highest levels of this vitamin. For these reasons, a good strategy to improve folate intake worldwide would be to genetically engineer common food plants to make more folates.

Hanson and Gregory’s laboratories (University of Florida’s Institute of Food and Agricultural Sciences) chose tomatoes (Solanum lycopersicum) for this experimental approach2,4. Their work is reviewed here.

Biochemistry of folates

Molecularly, folate is comprised of three parts—a p-aminobenzoate (PABA) central motif linked via its carboxyl group to one or more glutamate residues and a pteridine ring bonded to PABA’s amino group. In plants, these molecules are produced in different parts of the cell. Pteridines are synthesized in the cytosol, whereas plastids produce PABA. These building blocks are then joined together inside mitochondria to give dihydropteroate (DHP), which is finally glutamylated to synthesize folates.

In nature, folates can have one-carbon units attached to their N5 and/or N10 positions at different oxidation levels. Some examples are the 5-formyl, 5-methyl, and 10-formyl derivatives. Alternatively,

dihydrofolate (DHF) and other dihydro-derivatives may form by oxidation of the pteridine ring1. Folic acid per se is absent in nature.

Genetic engineering of folate biosynthesis Pteridine overproduction Díaz de la Garza and colleagues initially targeted the first step in pteridine biosynthesis to engineer increased folate production. This reaction is catalyzed by guanosine triphosphate cyclohydrolase I (GCHI) (Fig. 1). Overexpression of this enzyme greatly increased the flux of pteridines in tomato fruit; however, the folate content was increased only 2 – 4 times. A limited supply of the other building blocks that constitute the folate molecule could be a limitation. Consistent with this idea was the observation that PABA pools in the engineered plants were very small4.

Enhancement of p-aminobenzoate synthesis

The limitation caused by small PABA pools in tomato was overcome by genetically engineering the first enzyme of PABA synthesis, aminodeoxychorismate synthase (ADCS) (Fig. 1). Tomatoes were transformed with the Arabidopsis ADCS coding sequence (AtADCS) (including its native chloroplast targeting peptide) under control of the fruit ripeningspecific E8 promoter. RNA levels, measured at different ripening stages in the AtADCS+ transformants, were highest in fruit at the red stage. In these transformants, the level of accumulated PABA (56 nmol/g fresh weight) was 19-fold greater than in the empty vector controls. Although a marked increase in PABA was obtained in these transformants.