If we can identify the rate-limiting step and isolate the target gene, overexpression of the rate-limiting enzyme should be useful for increasing the accumulation of the desired compounds. The pioneering work by Yun et al. (1992) is an example of such a successful application in scopolamine biosynthesis in transgenic Atropa with Hyoscyamus H6H. The crucial point is identification of the rate-limiting step. While the early step is speculated to be rate-limiting for the whole pathway, simple overexpression of the early step enzyme into secondary metabolism is usually not sufficient for inducing the biosynthesis of the desired end-product due to a lack of sufficient induction of late-step enzymes. For example, the genes encoding TDC and STR have been extensively studied in C. roseus cell cultures. Overexpression of TDC resulted in higher levels of the immediate product tryptamine, but not increased levels of alkaloids; for STR, higher levels of alkaloids were noted (Canel et al., 1998). Administering such cell lines with tryptophan and terpenoid intermediates showed that they have the capacity for high alkaloid production, indicating that the terpenoid branch of the pathway is limiting (Canel et al., 1998; Whitmer et al., 1998). Such studies indicate that there are multiple rate-limiting processes. Accumulating results suggest that overall and integrated regulation of the biosynthetic pathway is crucial.
Other studies have also been aimed at increasing the flux through the biosynthetic pathway for both the tropane alkaloids and nicotine (Sato et al., 2001). Thus, the tobacco PMT gene was overexpressed in A. belladonna and N. sylvestris. Although a modest increase in PMT activity of up to 3.3-fold was found in transgenic A. belladonna plants, no increase in alkaloid levels was observed and only the levels of methyl putrescine was increased. In some transgenic N. sylvestris plants, PMT activity was increased four- to eightfold, whereas cosuppression was noted in other plants. The PMT-overexpressing transgenic lines showed a 40% increase in nicotine levels, whereas it was only 3% of that in the wild type in the case of cosuppression (Sato et al., 2001). A certain step in a pathway might appear to be rate-limiting, but overexpression of the encoding gene will, in most cases, immediately reveal new rate-limiting steps (Verpoorte and Memelink, 2002). These results are in contrast to those seen in overexpression of the gene encoding deoxyxylulose phosphate reductoisomerase in mint, which resulted in plants that had a normal phenotype and an almost 50% increase in essential oil (monoterpenoid) production (Mahmoud and Croteau, 2002, 2001).
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