Genetic Engineering of Tropane Alkaloid Biosynthetic Pathways
The earliest examples of engineering an alkaloid pathway in a medicinal plant/
plant tissue culture come from the tropane alkaloid field. The commercial source
of scopolamine is
Duboisia, originally cultivated in Australia.
A. belladonna accumulates
hyoscyamine rather than scopolamine as the major alkaloid. The cDNA
encoding
h6h from
H. niger was introduced into
A. belladonna using either
A. tumefaciens- or
A. rhizogenes-mediated transformation. Elevated levels of scopolamine
accumulated in the transgenic plants (up to 1.2% dry weight compared
to trace levels in control plants) and 0.3% in hairy roots compared to 0.03% in
control roots (Yun
et al., 1992). Since these original experiments, the tobacco
pmt has been expressed under transcriptional control of the dual CaMV 35S promoter
in
A. belladonna (Sato
et al., 2001). Regardless of the transgene transcript level,
the plants were phenotypically normal and had levels of hyoscyamine comparable
to control plants. The alkaloid profiles of transgenic hairy root lines were also quantitatively and qualitatively similar to wild type. In contrast, N. sylvestris
overexpressing the tobacco
pmt showed increases in leaf nicotine of up to 40%
compared to wild type (Sato
et al., 2001). Cosuppressed plants accumulated only
2% of wild-type nicotine levels and contained elevated levels of putrescine and
spermidine. Cosuppressed plants also showed phenotypic abnormalities.
The tobacco
pmt under transcritional control of the CaMV 35S promoter has
also been introduced into a scopolamine-rich
Duboisia hybrid (Moyano
et al., 2002). The N-methylputrescine levels of the resultant hairy roots increased twoto
fourfold compared to wild type, but levels of tropane alkaloids were not
increased. When the tobacco
pmt gene was introduced into
Datura metel and
Hyoscyamus muticus, the resultant hairy roots showed improved hyoscyamine
and scopolamine production in
D. metel, but only hyoscyamine content increased
in
H.
muticus (Moyano
et al., 2003). A similar
pmt-construct has been introduced
into
A. belladonna (Rothe
et al., 2003). Transgenic plants and derived root cultures
showed alkaloid profiles similar to those of controls. In summary, the expression
level of
pmt is apparently not rate limiting in all tropane alkaloid-producing
species.
Efforts have also been made to establish particle bombardment-mediated
transformation of
H. muticus to facilitate future metabolic engineering of this
species (Zeef
et al., 2000). Importantly in these experiments, fertile, mature plants
were recovered that could be self-pollinated.