Identification and characterization of key regulators of Paclitaxel Biosynthesis in Taxus cuspidata
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Date
28/06/2014Item status
Restricted AccessEmbargo end date
31/12/2100Author
Amir, Rabia
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Abstract
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.