Paclitaxel plants routes in bacteria

Abstract

U.S. and Singaporean researchers have engineered strains of Escheri-chia coli that produce two precursors of the cancer drug Taxol paclitaxel at up to 100-fold higher yields than previously attained in microorganisms. 1 The results suggest that complete synthesis of paclitaxel in bacteria is feasible and provide a shortcut that could help generate new bioactive analogs derived from the precursors taxadiene and taxadien-5α-ol. Paclitaxel was isolated from the Pacific yew tree (Taxus brevifolia) and is now produced via chemical reaction sequences consisting of 35 or more steps that result in overall paclitaxel yields of less than 1%. There is a shorter reaction sequence that starts with 10-deacetylbaccatin III, a paclitaxel precursor isolated from the European yew tree (T. baccata), but that reaction depends on a plant source for starting material. The inefficiency of both approaches has driven a continuing search for new ways to produce the drug. One alternative to chemical synthesis is biosynthesis in microorgan-isms (see Figure 1, " On the road to paclitaxel "). Such approaches tap the natural capacity of bacteria and yeast to produce isoprenoids—a class of five-carbon molecules that are building blocks for more complex molecules—and involve engineering the microorganism to express plant enzymes that assemble the isoprenoids into desired structures. 2–4 However, attempts to engineer E. coli and Saccharomyces cer-evisiae to produce taxadiene—the core hydrocarbon precursor of paclitaxel—have resulted in yields too low to be useful for subsequent chemical reactions. 5,6 The U.S.-Singapore team, led by Gregory Stephanopoulos and Blaine Pfeifer, hypothesized that the lackluster taxadiene yields in bacteria and yeast were caused in part by cellular toxicity of inter-mediates, adverse effects of the vectors used to increase gene expres-sion, and unknown pathways and molecules that inhibited taxadiene production. To overcome those obstacles, the team performed a combinato-rial analysis to probe how different promoter strengths and differ-ent copy numbers of genes encoding four bacterial and two plant enzymes affected taxadiene production in a total of 32 engineered strains of E. coli. The results of the analysis allowed the team to engineer E. coli expressing a specific combination of the six genes that produced taxadiene in yields that were about 100-fold higher than those previ-ously attained in bacteria or yeast. The incorporation of a third plant enzyme—T. brevifolia cyto-chrome P450 (p450)—into the bacteria enabled the oxidation of taxadiene into taxadien-5α-ol at yields that could be used for further chemical reactions, Stephanopoulos told SciBX. Stephanopoulos is a professor of chemical engineering and bio-technology at the Massachusetts Institute of Technology (MIT), and Pfeifer is an assistant professor of chemical and biological engineer-ing at Tufts University.