Select: Manipulating Cellular Machinery

Taxol (paclitaxel) is a potent anticancer therapeutic typically used to treat breast, lung, and ovarian cancers. Historically it has been produced by synthetic or semisynthetic routes, requiring as many as 51 synthesis steps or the use of a plant-derived intermediate, baccatin III, respectively. Ajikumar et al. (2010) introduce a new approach to produce Taxol: engineering the isoprenoid pathway in Escherichia coli. There are two pathways to consider for Taxol biosynthesis: the native upstream isoprenoid pathway produces metabolic precursors, and the downstream taxadiene pathway has been reconstructed successfully in E. coli. To date, most groups have assumed that these pathways are additive, without paying due attention to toxic side effects of intermediate metabolites or competing pathways and metabolites that could inhibit the drug production. With traditional combinatorial approaches unavailable for taxadiene, the team undertook a ‘‘multivariate modular pathway engineering’’ approach, splitting the pathway into two individually addressable modules. The upstream module focused on the four rate-limiting genes of the methylerythritol phosphate pathway. The downstream module comprised the two-gene pathway, resulting in taxadiene. By tweaking various genetic components and expression levels in the modules, the authors were able to optimize taxadiene production to levels 15,000-fold greater than the control, yielding 1.02 g/l (to date, the highest reported titers from metabolically engineered microbes have been a modest 10 mg/l). These yields notwithstanding, the key challenge in the generation of Taxolproducing microbes is the oxidation of taxadiene to taxadien-5a-ol via CYP450, a plant enzyme that is difficult to express in E. coli. By generating a chimeric CYP450, the authors are able to produce the alcohol but discover drastic reductions in taxadiene production, with concomitant increases in a pathway inhibitor, indole. Improvements to the CYP450-based oxidation chemistry that converts taxadiene to taxadien-5a-ol will open the door to commercial-scale synthesis of Taxol and other high-value terpenoids for use as chemicals or fuels. P.K. Ajikumar et al. (2010). Science 343, 70-74.