Taxol and β-tubulins from endophytic fungi isolated from the Himalayan Yew, Taxus wallichiana Zucc.

Abstract

Paclitaxel, better known as the anticancer drug Taxol®, has been isolated from several plant species and has been shown to be produced by fungi, actinomycetes, and even bacteria isolated from marine macroalgae. Given its cytostatic effect, studies conducted in the 1990's showed that paclitaxel was toxic to many pathogenic fungi and oomycetes. Further studies led to the idea that the differences in paclitaxel sensitivity exhibited by different fungi were due to differences in the β-tubulin protein sequence. With the recent isolation of endophytic fungi from the leaves and bark of the Himalayan Yew, Taxus wallichiana Zucc., and the availability of genomes from paclitaxel-producing fungi, we decided to further explore the idea that endophytic fungi isolated from Yews should be well-adapted to their environment by encoding β-tubulin proteins that are insensitive to paclitaxel. Our results found evidence of episodic positive/diversifying selection at 10 sites (default p-value threshold of 0.1) in the β-tubulin sequences, corresponding to codon positions 33, 55, 172, 218, 279, 335, 359, 362, 379, and 406. Four of these positions (i.e., 172, 279, 359, and 362) have been implicated in the binding of paclitaxel by β-tubulin or formed part of the binding pocket. As expected, all the fungal endophytes grew in different media regardless of the paclitaxel concentration tested. Furthermore, our results also showed that Taxomyces andreanae CBS 279.92, the first fungus shown to produce paclitaxel, is a Basidiomycete fungus as the two beta tubulins encoded by the fungus clustered together with other Basidiomycete fungi.

Paclitaxel – a Product of Fungal Secondary Metabolism or an Artefact?#

“…The underlying study combined methods of genome mining with morphological studies, based on genome sequence data obtained from the study of Heinig et al [60] and type specimen that had been deposited by Strobel et al [37] in the Farlow Herbarium at Harvard University. The study revealed that the fungus in question isas confirmed by another source [62] a wood-destroying basidiomycete, which makes the hypothesis of Taxol production in other fungi from the Ascomycota division appear even less plausible in terms of evolutionary biology [71]. The study concluded that contamination through the primary extraction of samples from bark or by Taxol residues in the fungi was the most likely explanation for the presumed aberrations of the Taxol hypothesis.…”

“…Also, no published efforts to reproduce the original research results could be observed. It is striking that the seminal publication by Heinig, Scholz, and Jennewein was mostly – and even in broad review articles – ignored 34 , 43 , 44 , 47 , 48 , 61 , 62 , 63 , sometimes dutifully cited, but nevertheless ignored in substance 33 , 44 , 64 . In some cases, the article is correctly summarized in one sentence, but the negative findings have not prompted the researchers to question their own assumptions or, at least, to discuss them in more detail 8 , 45 , 48 .…”

“…In this case, however, they could also save the energy to synthesize their own anti-mitosis agent. One article investigates the extent to which a different protein sequence in the β-tubulin of endobiotic fungi could lead to a relative insensitivity to the mitosis inhibitor Taxol 62 . This may explain how fungi survive in a toxic environment of yew trees, but it would not be proof that they can produce Taxol themselves.…”

“…Unfortunately, such scientific ethos of constructive skepticism and a willingness to also publish (supposedly) negative results are far too rare – in general and very much to the detriment of science. In the few articles that have appeared since then on Taxol biosynthesis, the contribution of the Stadler team is usually overlooked 34 , 62 , 72 and has, at least so far, only been recognized in one publication. The article was cited in passing in a table on sequencing that had taken place, but the negative findings have not yet been recognized or discussed 33 .…”

Use of medicinal plants as a remedy against lymphatic filariasis: Current status and future prospect

“…An example is found with case of the Yew tree that is the sources of the vital cancer drug taxol. Several endophytic fungi isolated from this tree have been shown to also produce taxol 51 . While the cultivation of tree plants is subject to long generational time, that of endophytic fungi is short and amenable to the culturing in fermenters which can be controlled to produce desirable quantities of useful compounds.…”