Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol

Abstract

Paclitaxel (Taxol) is a widely used anticancer isoprenoid produced by the secondary metabolism of yew (Taxus sp.) trees. However, only limited amounts of Taxol or related metabolites (taxoids) can be obtained from the currently available sources. In this work we have taken the first step toward genetically engineering the biosynthesis of taxoids in angiosperms. The first committed step in Taxol biosynthesis is the production of taxadiene from geranylgeranyl diphosphate (GGPP), catalyzed by the plastid-localized enzyme taxadiene synthase (TXS). A recombinant T. baccata TXS lacking the putative plastid targeting peptide and fused to a C-terminal histidine (His) tag was shown to be enzymatically active in Escherichia coli. Constitutive production of the full-length His-tagged enzyme in Arabidopsis thaliana plants led to the accumulation of taxadiene and concomitant growth retardation and decreased levels of photosynthetic pigment in transgenic plants. Although these phenotypes may derive from a toxic effect of taxadiene, the lower accumulation of endogenous plastid isoprenoid products such as carotenoids and chlorophylls in transgenic plants also suggests that the constitutive production of an active TXS enzyme might alter the balance of the GGPP pool. Induction of transgene expression using a glucocorticoid-mediated system consistently resulted in a more efficient recruitment of GGPP for the production of taxadiene, which reached levels 30-fold higher than those in plants constitutively expressing the transgene. This accomplishment illustrates the possibility of engineering the production of taxoids and other GGPP-derived isoprenoids in crop plants despite the constraints associated with limited knowledge with regard to regulation of GGPP availability.

Production of taxadiene from cultured ginseng roots transformed with taxadiene synthase gene

“…In contrast to the reduced growth shown in the TS-transformed Arabidopsis and tomato (29,31), no phenotypic or growth differences were observed between the cultured wild-type and the transgenic ginseng roots. Presumably, the newly introduced TS gene in Arabidopsis and tomato might convert GGPP to taxadiene, and therefore lower the amount of endogenous gibberellin (31).…”

“…Taxadiene is synthesized directly from GGPP found in most plant tissues, which serves as a common precursor for many metabolites in the terpenoid pathway. MJ increases terpenoid metabolites (24,31), and indeed, MJ treatment increased the yield of taxadiene in the cultured TS-transgenic ginseng roots, increasing by 1.6 times following only a 3-day MJ treatment (Fig. 4B).…”

“…Several studies have indicated methods to taxadiene in tomato and Arabidopsis using metabolic engineering due to the high price (29)(30)(31). The presence and expression of the TS gene in the transgenic cultured ginseng root and the de novo formation of a compound with the same fragmentation pattern as taxadiene confirmed the successful transformation of cultured ginseng roots to produce taxadiene.…”

“…Presumably, the newly introduced TS gene in Arabidopsis and tomato might convert GGPP to taxadiene, and therefore lower the amount of endogenous gibberellin (31). The moss engineered to produce taxadiene had no growth-retardation (30), because mosses do not require gibberellins for growth (32).…”

Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites

“…It led to accumulation of taxadiene in Arabidopsis cells [60]. This experiment demon strated the perspective of approaches based on engaging of natural terpenoid precursors of plant host in taxane biosynthesis paythway.…”