It is clear from some reports that in many cases a single gene will be insufficient to achieve high-level production of desired chemicals. For example, although transgenic lines overexpressing the gene encoding HPPD showed large increases in enzyme activity compared with control lines, only small increases in tocopherol content were observed (10% in leaves and 30% in seeds) [59, 60], suggesting that HPPD alone is not sufficient to increase tocopherol biosynthetic flux. Therefore, multipoint metabolic engineering is now beginning to supersede single-point engineering as the best way to manipulate metabolic flux in transgenic plants. Several points in a given metabolic pathway can be controlled simultaneously either by overexpressing and/or suppressing several enzymes, or through the use of tran-scriptional regulators to control several endogenous genes. Many researchers are fast approaching a time when sophisticated strategies for the multipoint manipulation of metabolic pathways can be modeled and implemented by multiple-gene transfer, thereby facilitating the production of desirable molecules in transgenic plants [101, 107].
Combined experiments on the co-overexpression of TyrA, HPPD and VTE2, HPT and y-TMT, VTE3 and VTE4 significantly elevated tocochromanol content or vitamin E activity to a level much higher than that derived from single-gene expression. Transcription factors offer great potential for the manipulation of metabolic pathways because of their ability to control both multiple-pathway steps and cellular processes that are necessary for metabolite accumulation , highlighting the potential benefit of using transcription factors to modify complex metabolic pathways in plants [109, 110]. The use of transcription factors to regulate vitamin E metabolic pathways in plants is still limited. No endogenous transcription regulators controlling the vitamin E pathway enzymes have been isolated. However, the potential of transcription factors to manipulate multiple fluxes of metabolic pathways will facilitate gene discovery and crop improvement.
Zhu et al.  overexpressed bacterial dihydrodipicohinate synthase in an Ara-bidopsis knockout mutant in the Lys catabolism pathway and affected a dramatic increase in free Lys in mutant. Their work elicitate us to design the overexpression of an enzyme resistant to tocopherol inhibition, together with the knock-out of the tocopherol catabolism pathway, and in order to produce much higher tocopherol levels than is produced by either strategy alone. On the other hand, we can break down the other metabolism pathways that consume common intermediates that are related to tocopherol synthesis.
Zeaxanthin is an important dietary carotenoid and can be converted to violax-anthin by zeaxanthin epoxidase. The gene for zeaxanthin epoxidase, with sense and antisense constructs, was transformed in potato (Solanum tuberosum). Both approaches (antisense and cosuppression) resulted in higher levels of zeaxanthin accumulation in potato tubers. In addition, a-tocopherol was elevated up to two- to threefold in the genetically transformed lines . This provides an example of the use of an antisense approach to knocking out one metabolic pathway gene to modulate the content of vitamin E.
The demonstration that data obtained from engineering tocopherol synthesis in model systems can be readily transferred to crop plants indicates that we are on the cusp of an exciting era in which plant metabolic engineering can be used to have a positive impact on human nutrition and health on a global scale.
The rationale is that, although most people can obtain sufficient amounts of vitamin E from a typical diet, current foods do not provide the therapeutic levels of vitamin E that would allow the public to enjoy the added health benefits of this vitamin. Biofortified plants would provide a sustainable alternative to a prescribed regimen of vitamin E supplementation that would be available to everyone the world over.
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