3.4.2. Heterologous expression
E. coli also serves as a heterologous host to produce taxadiene,
the key intermediate of taxol. In vitro synthesis of taxadiene in
genetically engineered E. coli with deoxyxylulose-5-phosphate
(DXP) overexpression produces 1.3 mg/l of taxadiene. This is an
alternative scheme to produce taxoids by non-taxol producing organisms
(Huang et al., 2001). Taxadiene synthesis
is divided into two pathways; precursor metabolism (upstream MEP
pathway) and taxadiene synthesis (downstream pathway). Induction of
pathway genes in K and B-derived E. coli strains triggered a
higher yield of taxadiene roughly 2.5-fold was obtained in K-derivative
as compared to B-derivative E. coli. It has also noted that
environmental conditions (temperature and varying exogenous
concentration of indole) influence taxadiene production in strains ofE. coli (Boghigian et al.,
2012).
Greater knowledge of the importance of biosynthetic pathway regulatory
genes can also be achieved by employing eukaryotic microbial
heterologous host. For example, the production of taxol biosynthetic
pathway precursor baccatin III in Saccharomyces cerevisiae using
episomal vectors resulted in increased taxol production by 100-fold
compared to Arabidopsis (Besumbes
et al., 2004; DeJong et al., 2006).
Co-overexpression of taxadiene synthase gene from T. chinensisand geranylgeranyl diphosphate synthase from Sulfolobus
acidocaldarius along with, mutant regulatory protein UPC2-1, resulting
in a 40-fold increase in taxadiene to 8.7 mg/l in the yeast cell
(Engels et al., 2008). Genetic
engineering of the taxol biosynthetic pathway in plant heterologous
hosts may also augment the accumulation of desired anticancer compounds.
For example, the insertion of the taxadiene synthase gene from theTaxus into Artemisia annua L. produced a high yield of
taxadiene in A. annua (129.7µg/g DW). It also resulted in the
accumulation of antimalarial compound artemisinin in the leaves of
Artemisia (Li et al., 2015). Likewise,
transformed roots of ginseng (Panax ginseng C.A. Meyer) harboring
a taxadiene synthase (TS ) gene from Taxus brevifoliaproduces 1 μg taxadiene per gram of dry weight. Furthermore, successful
accumulation of taxadiene in transgenic ginseng root is irrespective of
any change in phenotype and growth differences as compared to wild type
(Cha et al., 2012). De novo synthesis of
taxadiene also reported by using Nicotiana benthamiana as a
heterologous host. Agrobacterium- mediated transformation ofNicotiana benthamiana containing the taxadiene synthase gene from
the Taxus produced 11µg taxadiene/g of dry weight
(Hasan et al., 2014).