Jasmonate-responsive expression of paclitaxel biosynthesis genes in Taxus cuspidata cultured cells is negatively regulated by the bHLH transcription factors TcJAMYC1, TcJAMYC2, and TcJAMYC4.

Sangram K Lenka,Rosemary A Boshar,Kham Vongpaseuth,Elsbeth L Walker,N Ezekiel Nims,Susan C Roberts

doi:10.3389/fpls.2015.00115

Sangram K Lenka, Rosemary A Boshar + Show 4 more

Open Access

https://doi.org/10.3389/fpls.2015.00115

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Abstract

Taxus cell suspension culture is a sustainable technology for the industrial production of paclitaxel (Taxol®), a highly modified diterpene anti-cancer agent. The methyl jasmonate (MJ)-mediated paclitaxel biosynthetic pathway is not fully characterized, making metabolic engineering efforts difficult. Here, promoters of seven genes (TASY, T5αH, DBAT, DBBT, PAM, BAPT, and DBTNBT), encoding enzymes of the paclitaxel biosynthetic pathway were isolated and used to drive MJ-inducible expression of a GUS reporter construct in transiently transformed Taxus cells, showing that elicitation of paclitaxel production by MJ is regulated at least in part at the level of transcription. The paclitaxel biosynthetic pathway promoters contained a large number of E-box sites (CANNTG), similar to the binding sites for the key MJ-inducible transcription factor AtMYC2 from Arabidopsis thaliana. Three MJ-inducible MYC transcription factors similar to AtMYC2 (TcJAMYC1, TcJAMYC2, and TcJAMYC4) were identified in Taxus. Transcriptional regulation of paclitaxel biosynthetic pathway promoters by transient over expression of TcJAMYC transcription factors indicated a negative rather than positive regulatory role of TcJAMYCs on paclitaxel biosynthetic gene expression.

Highlights

Paclitaxel (Taxol®; Figure 1) is a diterpene derived from plants in the genus Taxus
ATG of the paclitaxel pathway gene are encoded as an N-terminal extension on the GUS protein. (B) The GUS TcJAMYC(s) full-length cDNA, and firefly luciferase (LUC) genes, all driven by the Cauliflower Mosaic Virus (CaMV) 35S promoter, that were used in bombardment assays
For each pathway gene promoter construct, GUS enzyme activity was at least 1.5fold higher in methyl jasmonate (MJ)-elicited cells compare to the control (Figure 3)

Summary

Introduction

Paclitaxel (Taxol®; Figure 1) is a diterpene derived from plants in the genus Taxus. Taxol triggers anti-mitotic and cytotoxic activity by disrupting normal tubulin dynamics leading to dysfunction of microtubules (Schiff et al, 1979). The biosynthetic pathway leading to paclitaxel has been only partially elucidated (Croteau et al, 2006; Ketchum et al, 2007; Long et al, 2008) (Figure 1), and improved understanding of the four or five undefined pathway steps as well as the overall regulation of paclitaxel synthesis are needed in order to enable bioengineering approaches. This will allow enhanced production of paclitaxel and potentially may allow production of novel bioactive taxanes in plant cells

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