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).