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).