Metabolic engineering of Nicotiana benthamiana for the increased production of taxadiene

Abstract

We report the production of taxadiene by transformation of N. benthamiana with a taxadiene synthase gene. The production was significantly increased by an elicitor treatment or metabolic pathway shunting. Paclitaxel (Taxol(®)) was first isolated from the bark of the pacific yew tree as an anticancer agent and has been used extensively to treat various types of cancer. Taxadiene, the first committed product of paclitaxel synthesis is cyclized from geranylgeranyl diphosphate (GGPP), and further complex hydroxylation and acylation processes of the unique taxane core skeleton produce paclitaxel. To accomplish de novo production of taxadiene, we transformed Nicotiana benthamiana with a taxadiene synthase (TS) gene. The introduced TS gene under the transcriptional control of the CaMV 35S promoter was constitutively expressed in N. benthamiana, and the de novo production of taxadiene was confirmed by mass spectroscopy profiling. Transformed N. benthamiana homozygous lines produced 11-27 μg taxadiene/g of dry weight. The highest taxadiene production line TSS-8 was further treated with an elicitor, methyl jasmonate, and metabolic pathway shunting by suppression of the phytoene synthase gene expression which resulted in accumulation of increased taxadiene accumulation by 1.4- or 1.9-fold, respectively. In summary, we report that the production of taxadiene in N. benthamiana was possible by the ectopic expression of the TS gene, and higher accumulation of taxadiene could be achieved by elicitor treatment or metabolic pathway shunting of the terpenoid pathway.

Plant Glandular Trichomes: Natural Cell Factories of High Biotechnological Interest

“…However, the amount produced is impressive compared with the 27 mg g dry weight obtained in the best stable transgenic N. benthamiana line (Hasan et al, 2014). Hasan et al (2014) could increase the production in N. benthamiana up to 48 mg g 21 dry weight by, in addition to constitutively overexpressing the taxadiene synthase, silencing the expression of the gene coding for the phytoene synthase and thereby increasing the geranylgeranyl diphosphate availability. In N. tabacum, silencing the genes coding for CBTS did not increase the production of taxadiene .…”

“…As a result, the MEP-derived primary metabolites were not synthesized in sufficient amounts to sustain normal growth. Only a few attempts led to the production of taxadiene in plants without any defect, as in ginseng (Panax ginseng; Cha et al, 2012) and N. benthamiana (Hasan et al, 2014) by the ubiquitous overexpression of the taxadiene synthase, but the amount produced was quite low.…”

Transgene Biocontainment Strategies for Molecular Farming

“…The genetic engineering of N. benthamiana to express a taxadiene synthase gene, which produces taxadiene from geranylgeranyl diphosphate (GGPP), produced 11-27 µg/g DW taxadiene. Further suppression of the phytoene synthase gene and addition of methyl jasmonate increased taxadiene accumulation to 35 µg/g DW (Hasan et al, 2014). The successful de novo production of taxadiene could lead to the development of a heterologous plant system that biosynthesises Taxol.…”

Plant Metabolic Engineering Strategies for the Production of Pharmaceutical Terpenoids

“…Hasan et al (2014) constitutively overexpressed the TbTS gene in N. benthamiana. Transformed N. benthamiana line produced 11–27 μg/g DW taxadiene.…”