Loake, Gary

Hudson, Andrew

Amir, Rabia

other

2016-09-12T10:47:28Z

2016-09-12T10:47:28Z

2014-06-28

Numerous drugs in the current pharmacopoeia originate from plant sources. Plant cell culture represents an alternative source for producing high-value secondary metabolites including paclitaxel. Paclitaxel is mainly derived from the plant genus Taxus and has been widely used in cancer chemotherapy. However, plant cell culture is often not commercially viable because of difficulties associated with culturing dedifferentiated plant cells (DDCs) on an industrial scale. Therefore, we isolated and cultured innately undifferentiated cambial meristematic cells (CMCs) from Taxus cuspidata, which possess superior growth properties relative to DDCs. These CMCs have been demonstrated to be a cost effective platform for the sustainable production of paclitaxel. Using 454 sequencing, we determined the transcriptome of T. cuspidata CMCs. Utilizing this transcriptome as a reference, we then employed Solexa digital gene expression profiling to identify transcriptional regulators that were induced by methyl jasmonate, an activator of paclitaxel biosynthesis. This lead to the discovery of 19 putative transcription factors (TFs) belonged to 5 TF families which were further confirmed by associated molecular methods. We aimed to identify which of these 19 regulatory proteins drive the expression of 5 paclitaxel biosynthetic genes by employing yeast one-hybrid analysis and electrophoretic mobility shift assays. Further, the cis-regulatory elements associated with these TFs were identified in the promoter regions of the two early, taxadiene synthase (TASY) and taxadiene 5α hydroxylase (T5αH), and three late, 10-deacetylbaccatin III-10-O-acetyltransferese (DBAT), phenylalanine aminomutase (PAM) and 3'-N-debenoyl-2-N-benzoyltransferase (DBTNBT), paclitaxel biosynthetic genes to facilitate the TF-DNA binding studies. Finally, understanding the TF regulatory network underlying paclitaxel biosynthesis can guide the engineering of CMCs to elevate the production of this key pharmaceutical.

http://hdl.handle.net/1842/16467

en

The University of Edinburgh

Lee, E. K., Jin, Y. W., Park, J. H., Yoo, Y. M., Hong, S. M., Amir, R., Yan, Z., Kwon, E., Elfick, A., Tomlinson, S., Halbritter, F., Waibel, T., Yun, B. W., and Loake, G. J. (2010). Cultured cambial meristematic cells as a source of plant natural products. Nature Biotechnology 28, 1213-1217.

Yun, B. W., Yan, Z., Amir, R., Hong, S., Jin, Y. W., Lee, E. K., and Loake, G. J. (2012). Plant natural products: history, limitations and the potential of cambial meristematic cells. Biotechnology and Genetic Engineering Reviews 28, 47-60.

2100-12-31

Paclitaxel

Taxus cuspidata

transcription factors

methyl jasmonate

Identification and characterization of key regulators of Paclitaxel Biosynthesis in Taxus cuspidata

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy

Restricted Access