An intron-free methyl jasmonate inducible geranylgeranyl diphosphate synthase gene from Taxus media and its functional identification in yeast

Geranylgeranyl diphosphate synthase (GGPPS) EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes and, in particular, Taxol, one of the most potent antitumor drugs. In order to investigate the role of GGPP synthase in Taxol biosynthesis, we cloned, characterized, and functionally expressed the GGPPS gene from Taxus media. Using the genome walking strategy, a 3743-bp genomic sequence of T. media was isolated which contained a 1182-bp open reading frame (ORF) encoding a 393-amino acid polypeptide that showed a close similarity to other plant GGPPSs. Subsequently, the full-length cDNA of the GGPPS gene of T. media (designated TmGGPPS) was amplified by RACE. Bioinformatic analysis showed that TmGGPPS was an intron-free gene, and its deduced polypeptide contained all five conserved domains and functional aspartate-rich motifs of the prenyltransferases. By constructing the phylogenetic tree of plant GGPPSs, it was found that plant-derived GGPPSs could be divided into two classes, those of angiosperms and gymnosperms, which might have evolved in parallel from the same ancestor. To our knowledge, this was the first report that the geranylgeranyl diphosphate synthase genes were free of introns and evolved in parallel in both angiosperms and gymnosperms. The coding sequence of TmGGPPS was expressed through functional complementation in a yeast mutant lacking GGPPS activity (SFNY368), and the transgenic yeast was shown to have this activity. This was also the first time SFNY368 was used to identify the function of plant-derived GGPPSs. Furthermore, investigation of the effect of methyl jasmonate (MeJA) on the expression of TmGGPPS showed that MeJA-treated T. media cultured cells had much higher expression of TmGGPPS than untreated cells.From Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 14–20.Original English Text Copyright © 2005 by Zhihua Liao, Yifu Gong, Guoyin Kai, Kaijing Zuo, Min Chen, Qiumin Tan, Yamin Wei, Liang Guo, Feng Tan, Xiaofen Sun, Kexuan Tang.This article was submitted by the authors in English.