Metabolic engineering of the diosgenin biosynthesis pathway in Trigonella foenum-graceum hairy root cultures

Diosgenin as a triterpene with numbers of pharmaceutical applications has been identified in Trigonella foenum-graceum. In this survey, in order to scale up the amount of diosgenin in Fenugreek as a promising alternative of yam, ∆²⁴-reductase as a rate limiting enzyme in diosgenin biosynthesis pathway has been overexpressed by utilizing pBI121 expression plasmid in hairy roots culture platform. The recombinant binary vector pBI121-∆²⁴-reductase was transformed into R. rhizogenes strain ATCC 15834 to induce transgenic hairy roots in "Hamedan" as a low-diosgenin production genotype. In the transgenic hairy roots, the ∆²⁴-reductase expression level was significantly 8.15 times overexpressed comparing to the non-transgenic hairy roots, Nonetheless the Sterol-methyltransferase, as a competitive enzyme, was 6 times downregulated. Furthermore, the expression rate of Squalene synthase, Cycloartenol synthase, C26-Hydroxylase were also increased 1.5, 1.7, 2.9 times higher than those of the non-transgenic hairy roots, respectively. The diosgenin content in the transgenic hairy root was raised 3 times up comparing to the non-transgenic hairy roots, besides it was scaled up 25-fold comparing to the diosgenin amount in "Hamedan" Leaf. As a result, the first metabolic engineering on this pathway was clearly revealed the impact of ∆²⁴ -reductase gene in diosgenin content enhancement.