Research Projects

The glandular trichomes (GTs) of Nicotiana tabacum are the impressive producers of diterpenoid class of secondary metabolites that are named as labdanoids and cembrenoids. High density of highly productive GTs in N. tabacum suggests that they are an attractive target for the metabolic engineering of economically and medicinally important diterpenoids. For the metabolic engineering, one of the major limitations is the availability of sufficient precursor pool for the engineered enzymes. In that regard, the GTs appear to have sufficient supply of GGPP (geranyl geranyl diphosphate) pool. The biosynthesis of glandular trichome-localized diterpenoids (labdanoids and cembrenoids) involves CBTS and CYP2, as the entry step enzymes, which act upon GGPP and lead to the biosynthesis of cembrenoids and labdanoids, respectively. Thus, the inactivation of these two enzymes might lead to the abolition of the production of labdanoids and cembrenoids in the GTs, and there will be an accumulation of GGPP (geranyl geranyl diphosphate), which could be channeled towards the biosynthesis of desired diterpenoids, through strategic genetic engineering of the genes involved in the pathway. With the above-mentioned hypothesis, in the present project proposal, we intend to modify the endogenous diterenoid biosynthesis of GTs of N.tabacum through genome editing tool. The loss of function mutations will be introduced in the CBTS and CYP2 genes through Crispr/Cas based genome editing. The developed knockout mutant of N. tabacum is expected to be devoid of endogenous trichome-specific diterpenoids. Thus, the GGPP pool will be available in sufficient amount for a heterologous enzyme, involved in the biosynthesis of other economically important diterpenoids. As a proof of concept, the knockout mutants of CBTS and CYP2 genes will be engineered with a terpene synthase, which is responsible for the biosynthesis of the precursor (taxadiene) for taxol biosynthesis using the GGPP pool in GTs. The terpene synthase, named as taxadiene synthase (TS) will be expressed under the control of a trichome-specific promoter in the mutant background of N. tabacum. The resultant transgenic lines are expected to produce substantial amount of taxadiene in the GTs, without having any growth defects. Taken together, the present proposal will develop tobacco GTs as a platform for the metabolic engineering of high-value diterpenoids.