Overexpression of a 10-deacetylbaccatin III-10 β-O-acetyltransferase gene leads to increased taxol yield in cells of Taxus chinensis

Abstract

Taxus chinensis suspension cells were transformed by the Agrobacterium-mediated transformation method to overexpress the dbat gene coding for 10-deacetylbaccatin III-10 b-O-acetyltransferase, a key enzyme for taxol biosynthesis. A. tumefaciens strain LBA4404 harboring either pCAMBIA1303 or the recombinant plasmid p1303-SdbatN was used. Both plasmids harbored the hygromycin phosphotransferase gene (hptII) and gusAmgfp5 gene as selectable markers, but the latter plasmid also harbored the dbat gene in the T-DNA region. The transgenic T. chinensis cells had been maintained in modified Gamborg's B5 medium supplemented with hygromycin for more than 14 months and were subcultured at 4-week intervals. The selected transgenic cells were identified by PCR, Southern blot analysis, b-glucuronidase assay and western blot analysis, and the results showed that the transgenes were integrated in the chromosomal DNA of T. chinensis cells with single-copy style, and that the gusAmgfp5 reporter gene was expressed in the transgenic cells. The dbat mRNA expression level in the p1303-SdbatNtransgenic T. chinensis cells tested by real-time quantitative PCR was 5.3 ± 0.6 times that of the non-transformed cells. Taxol yield of the p1303-SdbatN-transgenic T. chinensis cells was about 1.7 times that of the non-transformed cells. These results suggest that the overexpression of dbat gene in transgenic T. chinensis cells leads to increased taxol yield.

A reliable protocol for the stable transformation of non-embryogenic cells cultures of grapevine (Vitis vinifera L.) and Taxus x media

“…Approximately 90-95% of Vitis and 75% of Taxus transformed calli were successfully maintained under continuous paromomycin selection. This high transformation efficiency is, as far as we know, the best ever reported since previous studies provided evidence of only the expression of reporter genes, but not of transformation efficiency [41,69]. Unlike the previous work by Gollop et al [41], the work described herein: (i) provides direct evidence for absence of contamination by Agrobacterium in transgenic cultures of V. vinifera, cv Gamay; (ii) skips the need to keep the culture under continuous exposure to cefataxime, except for the first selection period; and (iii) skips the need to use antioxidants to counteract the stress of the cell culture in any stage of the transformation and selection process.…”

Changes in gene transcription and taxane production in elicited cell cultures of Taxus×media and Taxus globosa

“…cell cultures (TCC) has been empirically optimized, from a small scale to bioreactor level (Cusidó et al, 2002;Bentebibel et al, 2005). Metabolic engineering techniques have also been employed to obtain cell cultures overexpressing genes of the taxol biosynthetic pathway (Exposito et al, 2010;Zhang et al, 2011). With the aim of exploring how the different taxane productionboosting factors also affect gene expression and metabolic profiles in TCC, several rational studies have focused on the molecular bioprocesses in the producer cells (Croteau et al, 2006;Nims et al, 2006;Onrubia et al, 2010Onrubia et al, , 2011Sabater-Jara et al, 2014).…”

Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis

“…However, the yield of taxol is low in cultured Taxus cells, which limits the application of cell culture (Walker & Croteau 2000). One effective approach is metabolic engineering of plant cells in an attempt to obtain higher taxol output (Roberts 2007; Zhang et al. 2011).…”