Paclitaxel is an antitumor diterpene from Taxus spp. that binds tubulin, stabilizes microtubules and induces apoptosis in dividing human cells. It was originally isolated from the bark of Taxus brevifolia and approved for clinic uses by the FDA in 1992. Because of its excellent activity in treatment of various cancers, a significant supply shortage has been created by the enormous demand for this natural product. Thus, researchers have been focusing on the development of effective ways to increase the production of paclitaxel and related bioactive molecules. This shortage was initially solved by over-harvesting of T. brevifolia bark; however, it is not an environment-friendly, effective and sustainable way to supply paclitaxel. A semisynthetic route was then developed to convert the more readily available and renewable 10-deacetylbacatin III into paclitaxel. As an alternative, plant cell cultures have been employed to commercially produce paclitaxel and it is a more environment-friendly and sustainable route to end the supply crisis. However, problems associated with plant cell culturing at an industrial scale, such as cell aggregation and variability in yield, significantly affect paclitaxel production. Therefore, a discovery of a better-performing Taxus cell line might be a solution to overcome these culturing-associated problems. A cambial meristematic cell (CMC) line of Taxus cuspidata has been isolated, cultured and demonstrated to be a cost-effective and environmentally friendly platform for the sustainable production of paclitaxel (Lee et al. 2010). Compared to dedifferentiated cell (DDC) lines, CMC lines are undifferentiated cells and proved to have stem cell-like properties. When cultured at an industrial scale, this cell line contains much smaller cell aggregates with many cells appearing as singletons, the biomass of which is still increasing after 22-month culturing, and has much greater paclitaxel production after elicitation (Lee et al. 2010). In my project, we aimed to identify the transcription factors (TFs) that regulate the expression of paclitaxel biosynthesis genes. We performed Illumina Solexa sequencing on cDNA libraries derived from methyl jasmonate (MeJA)-elicitated CMCs to digitally profile gene expression. Analysis of differentially expressed gene (DEG) abundance led to the discovery of 19 putative TFs and bioinformatic analysis further showed that these 19 TFs belong to 5 different TF families. Further, the DNA binding motifs associated with these TFs can be found in the promoters of the two early, taxadiene synthase (TASY) and taxadiene 5α hydroxylase (T5αH), and three late, 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT), phenylpropanoyltransferase (PAM) and 3’-N-debenzoyl-2-deoxytaxol-Nbenzoyltransferase (DBTNBT), paclitaxel biosynthesis pathway genes. Then, yeast one-hybrid analysis, gel shifting assays and plant transient expression assays (TEA) were employed to assay TFs that interact with these promoters. Although Y1H screening did not show any convincing TF-promoter interactions, the attempted plant transient expression assay in the leaves of Nicotiana benthamiana might be a more suitable system to screen the positive regulators. Finally, the elucidation of a TF regulatory network that controls paclitaxel biosynthesis will guide the rational engineering of CMCs to ultimately increase yields of this important pharmaceutical.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:669321 |
| Date | January 2013 |
| Creators | Yan, Zejun Jun |
| Contributors | Loake, Gary; French, Chris |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11716 |
Page generated in 0.0019 seconds