Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Molecular Biotechnology of Microbiological Systems
Plant Genetic Engineering: Applications
Modification of the Nutritional Value of Plants

Over the years, agronomists and plant breeders have made significant progress in improving the quality and increasing the yields of A wide variety of agricultural crops. However, Traditional Methods of developing new plant varieties through crossbreeding are very labor-intensive, time-consuming, and limited in their potential due to the restricted Gene pool available within crossed lines. Introduction/32.html">Genetic Engineering techniques not only accelerate The process of obtaining plants with enhanced traits but also make it possible to create varieties with novel characteristics that could never be transferred via traditional crossbreeding. For example, laboratory-derived crops with improved nutritional qualities, such as maize and peas, have already been successfully produced. In these plants, the Amino Acid Composition of certain seed storage Proteins was modified. Furthermore, oilseed varieties (both edible and non-edible) with altered fruit fatty acid profiles have been developed, and attempts have even been made to improve fruit flavor by introducing the gene for monellin—a sweet-tasting protein—into plants.

Amino Acids

Storage proteins, which serve as carbon and nitrogen sources for germinating seeds, consist of a limited, repetitive set of amino acids. The Nutritional Value of these proteins is low because they lack one or more Essential Amino Acids (typically Lysine or Methionine). While The amino acid composition of seed storage Proteins can be slightly modified through conventional crossbreeding, Genetic engineering METHODS have recently been applied for this purpose.

In one preliminary experiment, the phaseolin gene from common beans—which encodes a storage protein composed of a diverse array of amino acids—was introduced into tobacco plants. The gene was efficiently expressed, and the protein product was successfully delivered to the appropriate cellular compartment. In addition, by specifically altering The nucleotide sequence of seed storage protein genes in vitro, it became feasible to synthesize proteins with a tailored amino acid composition. When Amino Acid Substitutions occur near the hypervariable region of the C-terminal domain, the overall Structure OF THE molecule remains undisturbed. Proper chain folding is also maintained during seed germination.

To increase the lysine content in seeds, an attempt was made to deregulate its Biosynthesis. The amino acids lysine, Threonine, methionine, and isoleucine are synthesized from aspartate (Fig. 18.16) in a multi-step pathway. The first step involves the phosphorylation of aspartate by aspartate kinase (AK) to yield ß-aspartyl phosphate. Subsequently, in the lysine biosynthetic pathway, the Condensation of aspartate ß-semialdehyde with Pyruvate is catalyzed by dihydrodipicolinate synthase (DHDPS). The regulation of both enzymatic activities (AK and DHDPS) is governed by lysine via feedback inhibition, a mechanism that must be circumvented to allow unrestricted lysine synthesis. To achieve this, DHDPS and AK genes insensitive to lysine inhibition were isolated from Corynebacterium and E. coli, respectively. Each of these genes was fused to a nucleotide sequence encoding a leader peptide that targets proteins to METABOLISM/14.html">Chloroplasts, equipped with a seed-specific promoter, and introduced into canola and soybean plants using a Ti-plasmid-based binary vector (Fig. 18.17). Seeds from the Transgenic Plants contained up to 100 times more free lysine than those of conventional plants; furthermore, the total lysine content doubled in all canola seed proteins and increased fivefold in soybean proteins.

When maize is used as livestock feed, it is typically supplemented with soybean meal and purified lysine. However, rather than using costly purified lysine, one could Supplement maize with inexpensive soybean meal derived from transgenic soybean lines that synthesize high levels of lysine. It is likely that this approach, successfully applied in soybeans, can also be used to develop a maize variety with an enhanced lysine content in its seeds, thereby significantly increasing its nutritional value.

Lipids

It is estimated that the global production of plant oils reached approximately $45 billion in 1995 and is projected to hit $70 billion by 2010. Over 90% of these oils are consumed in The production of margarine, shortening, salad dressings, and cooking oils. Roughly 75% of all oilseed crops comprise soybean, oil palm, rapeseed (canola), and sunflower, and the oils derived from them consist primarily of the following Fatty acids: palmitic, stearic, oleic, linoleic, and linolenic acids (Table 18.5).

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Fig. 18.16. Schematic pathway of aspartate-derived Amino acid biosynthesis (not all reactions and intermediates are shown). Dashed arrows indicate feedback inhibition. DHDPS stands for dihydrodipicolinate synthase; AK stands for aspartate kinase.

Fig. 18.17. Ti-plasmid vector used for the transformation of soybean and canola to increase their lysine content. Pv5' is the promoter of the bean ß-phaseolin gene, Pv3' is the Transcription termination signal of the bean ß-phaseolin gene, cts is the sequence encoding the chloroplast transit peptide of the ribulose-1,5-bisphosphate carboxylase small subunit, dapA is the Corynebacterium gene encoding a lysine-insensitive dihydrodipicolinate synthase, lysCM4 is the mutant E. coli lysC gene encoding a lysine-insensitive aspartate kinase, and L and R denote the left and right T-DNA border sequences, respectively.

Genetic engineering makes it possible to alter the degree of unsaturation (i.e., the number of C=C double bonds) and the carbon chain length of these fatty acids. Numerous transgenic canola varieties that synthesize oils with modified fatty acid profiles have been developed and field-tested (Table 18.6). Each transgenic variety carried a single additional gene. For example, plants engineered to synthesize high levels of stearic acid carried an antisense copy of the Brassica stearoyl-ACP desaturase gene, which suppressed the expression of the endogenous canola gene and led to the accumulation of stearic acid—a compound normally converted into oleic acid. The success achieved in generating transgenic canola varieties offers great promise that this approach will find widespread commercial application in the future to create novel, commercially valuable crop varieties.

Table 18.5. Some important plant fatty acids

¹⁾ The first number in the subscript following C indicates the number of carbon atoms, and the second indicates the degree of unsaturation, i.e., the number of C=C double bonds; the symbol ∆ followed by a number denotes THE POSITION OF the first carbon atom forming a C=C double bond, and the number preceding OH specifies the position of the side hydroxyl group. All C=C bonds are in the cis configuration. In standard Fatty Acid Nomenclature, carbon number 1 is assigned to the carboxyl carbon.