Metabolic engineers have access to a vast array of molecular and genetic tools to rewire plant metabolism, most of which aims at the modulation of enzyme activity either toward an increase or a decrease of metabolic flux through a given pathway. In the simplest case a single enzymatic step is the target for modulation. To increase the production of a desired compound or a novel compound, genes encoding biosynthetic enzymes of the pathway can be overexpressed. Further increases in flux can be achieved by overexpressing enzymes from heterologous sources which are not subject to regulation or which have different regulatory properties compared to the endogenous plant enzyme. A problem associated with overexpression of single enzymes is that other steps in the pathway can become limiting and thus total metabolic flux does not substantially increase. To circumvent this, several consecutive enzymes in the same pathway must be up-regulated at the same time, either by transferring several expression cassettes into the plant or by overexpression of regulatory proteins, i.e. transcription factors. The latter approach, however, requires transcriptional co-regulation of all steps in a pathway as it has been shown for a number of pathways in plant secondary metabolism (Broun 2004; Grotewold 2008).
To reduce the levels of undesirable gene products, two general approaches are commonly used: recessive gene disruption and dominant gene silencing. In gene disruption approaches, the target sequence is mutated to eliminate a particular gene function, whereas dominant gene silencing methods induce either the destruction of the gene transcript or the inhibition of transcription. So far, directed gene disruption is not efficient in higher plants. Therefore, the most widely used technologies for the generation of loss-of-function mutants are transposon mutagenesis (Altmann et al. 1995; Kim et al. 2004), Agrobacterium T-DNA insertions (An et al. 2003; Jeon et al. 2000; see also Chap. 1) and, more recently, the use of chemical mutagenesis in combination with TILLING (Henikoff et al. 2004) to create disruptions in coding regions of genes. These techniques have been proven very useful for functional genomics; however, their use for metabolic engineering is limited. They are restricted to a few genetically tractable plant species and due to their untargeted nature they require the generation of large populations of mutated plants to screen for a desired mutation. In addition, genetic redundancy caused by multi-gene families and polyploidy further complicates these kinds of knock-out approaches.
RNA interference (RNAi) and related mechanisms such as 'antisense' or 'co-suppression' are homology-dependent gene-silencing technologies that possess a great potential for metabolic engineering (Mansoor et al. 2006; Tang et al. 2007; see Chap. 5 for details on the mechanism). In comparison with gene disruption, RNA silencing bears several advantages. It is a dominant trait that can be introduced into any transformable plant species, including the transfer into elite crop varieties. Owing to its targeted nature it does not require the generation of overly large populations of transgenic plants to find a suitable event. Especially in the case of antisense or co-suppression, the efficiency of silencing can vary considerably between individual transformants. This allows the manipulation of metabolic steps where a loss-of-function would be detrimental to the plant but a decrease of gene expression at 30-90% yields a desired metabolic phenotype. Furthermore, by the use of specific promoters, RNA silencing can be manipulated in a spatial and temporal manner. This is important for genes where down-regulation is good for the improvement of a specific organ, e.g. seeds or tubers, but is deleterious to the growth of other plant organs.
Examples are provided below to show how the above strategies are applied to manipulate the production of different classes of compounds.
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