Getting to the bottom of Taxol biosynthesis by fungi

Uwe Heinig Susanne Scholz Stefan Jennewein 0 ) Fraunhofer Institut fr Molekularbiologie und Angewandte kologie , Forckenbeckstrasse 6, 52074 Aachen, Germany Taxol (paclitaxel) is a highly-oxygenated diterpenoid natural product first isolated from the pacific yew tree (Taxus brevifolia). It is one of the most widely used anticancer drugs. Soon after the discovery of its unique mode of action and the resulting high demand, an extensive search was initiated for alternative sources to replace the slow-growing and scarce pacific yew. Thus far, however, Taxol and related compounds have only been found in the genus Taxus, which comprises a small number of slow-growing plants with a broad but generally isolated geographical distribution. In 1993, Stierle and colleagues reported the independent biosynthesis of Taxol in an endophytic fungus isolated from Taxus brevifolia, which resulted in more than 160 subsequent publications and patents addressing the biosynthesis of Taxol and related taxanes by microorganisms. The literature on fungal taxane synthesis contains numerous inconsistencies, prompting us to thoroughly re-examine Taxol biosynthesis in endophytic fungi associated with Taxus spp. Using a combination of phytochemistry, molecular biology and genome sequencing, we were unable to find any evidence for independent taxane biosynthesis in any of the endophytes, including the isolate described in the original publication (Taxomyces andreanae) and several more recent isolates from Taxus trees. Our findings therefore resolve a long-standing mystery concerning the evolution of a complex terpenoid biosynthetic pathway in two distantly-related organisms. - Taxol (paclitaxel), a highly-oxygenated diterpenoid natural product first isolated from the pacific yew tree (Taxus brevifolia), is arguably one of the most successful anticancer drugs of all time (Suffness and Wall 1995; Brown 2003). The limited supply of Taxol and related compounds made pharmaceutical development a major challenge (Suffness and Wall 1995). Therefore, soon after its unique mode of action was discovered, an extensive search was launched to find alternative sources because the pacific yew is slow-growing and scarce (Croom 1995; Itokawa 2003). For a long time, Taxol biosynthesis was thought to be restricted to the ancient Taxus genus (Taxaceae, Coniferales), which comprises 11 geographically-isolated species. Fossil records indicate that yew trees have existed for more than 200 million years with little evolutionary change. Taxus grandis from the Quaternary period shared many characteristics with the modern yew, Taxus baccata (Croom 1995). Considering the age and isolation of the genus together with the extreme longevity of individual members (some yew trees live more than 3,000 years), it was believed that the Taxol metabolic pathway was unique to this genus. Members of the closely related genera Pseudotaxus and Austrotaxus do not synthesize Taxol, although simple taxanes lacking the oxetane or Dring structure have been isolated from Austrotaxus spicata, the only member of the genus Austrotaxus, which is regarded as a primitive ancestor of Taxus (Guritte-Voegelein et al. 1987). Pseudotaxus spp. do not produce taxanes at all. The evolutionary advantage of Taxol biosynthesis in yew trees remains a mystery, particularly in light of the production of the highly cardiotoxic but chemically less complex taxines by several species. More than 360 taxanes have been identified in different Taxus spp. (Baloglu and Kingston 1999; Itokawa 2003) but Taxol (if present at all) represents only a minor fraction of the total taxane complement. The biosynthesis of Taxol and other taxanes is well characterized (Croteau et al. 2006; Kaspera and Croteau 2006; Heinig and Jennewein 2009) and appears to follow an anastamosing pattern that yields several physiologically-active products as well as metabolic dead ends (Fig. 1). Several of the key steps involved in the 20 or more enzymatic reactions required to produce Taxol have been characterized at the biochemical and genetic levels (Croteau et al. 2006; Jennewein et al. 2004b). The biosynthetic pathway, starting with the cyclization of geranylgeranyl diphosphate to form taxa-4(5),11(12)-diene, involves enzymes from several different classes that are located in several different cellular compartments, including the plastid, endoplasmic reticulum and cytosol. In 1993, Stierle and colleagues reported the unprecedented isolation of a Taxus spp. endopyhtic fungus (Taxomyces andreanae) that could synthesize Taxol and other taxanes such as baccatin III independently. This was demonstrated using radiolabeled precursors, such as 14C-phenylalanine and 14C-acetate (Stierle et al. 1993). Even more surprisingly, Taxol compromised an unusually high percentage (1520 %) of the total taxane fraction synthesized by the fungus compared to that synthesized by the yew. The isolated Taxomyces andreanae was subject to a patent application and d (...truncated)