mechanism of cyclization. Incubation of (R)-[4-2H1]geranylgeranyl diphosphate gives a 10:10:80 mixture of [5beta-2H1]taxa-3(4),11(12)-diens, [5beta-2H1]taxa-4(20),11(12)-diene and unlabeled taxa-4(5),11(12)-diene
exogenous Taxol (paclitaxel) causes an considerable increase in taxadiene synthase activity, the expression level peaks at day 4 with a rapid decrease at day 8, reaching a second, lower, peak at day 12. By day 16, ts mRNA is only detectable in the untreated control at a very low level. The concentration of taxol that most effectively induces ts mRNA expression is 50 mg/l compared to the control, the transcript levels are 2.5-, 3.1- and 3.3fold higher at 4, 8 and 12 days of culture.
coexpression of geranylgeranyl diphosphate synthase gene from Sulfolobus acidocaldarius and codon optimzing of the Taxus chinensis taxadiene synthase gene increases the production of taxadiene in Saccharomyces cerevisiae
a relatively high level of activity is observed at day 4, when it is 2.8-, 6.8-, 3.2- and 1.7fold higher, respectively, in the cultures treated with 25, 50, 100 and 200 mg/l taxol than in the untreated control (6.64 pkat/mg of protein). Enzyme activity then decreases markedly from days 4 to 12 and thereafter is measurable only at a very low level until day 20.
Replacement of the geranylgeranyl diphosphate synthase with the enzyme from Sulfolobus acidocaldarius and codon optimizing of the Taxus chinensis taxadiene synthase gene to ensure high-level expression, resulting in a 40fold increase in taxadiene to 8.770.85 mg/l as well as significant amounts of geranylgeraniol (33.1 mg/l)
detailed mechanistic analysis of the biosynthesis of taxadiene by enzyme TXS using a hybrid quantum mechanics-molecular mechanics potential in conjunction with free energy simulation methods, overview. Generation of a free-energy fingerprint for type I terpene synthases. The active-site Trp residue W753 is a key feature of the TXS active-site architecture and stabilizes intermediate cations via Pi-cation interactions
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X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N-terminus, complexed with 13-aza-13,14-dihydrocopalyl diphosphate, at 1.82 A resolution, and 2-fluorogeranylgeranyl diphosphate, at 2.25 A resolution. The structure reveals a modular assembly of three alpha-helical domains. The C-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third insertion domain together adopt the fold of a vestigial class II terpenoid cyclase
the mutation leads to the exclusive formation of side product cembrene A. The simulations of the W753H mutant shows that, in the mutant structure, the His side chain is in the perfect position to deprotonate the cembrenyl cation en route to cembrene formation and that this abortive deprotonation is an energetically facile process
cDNA is directly cloned into the pGemT-Easy vector, sequenced and cloned into the plant binary transformation vector pBC35. Furthermore it is subcloned into pBluescript, released and ligated into pRD12. The pBC35 and pRD12 constructs are transferred to Agrobacterium tumefaciens and used to transform tomato cotyledons
Expression of Taxus chinensis taxadiene synthase (expression of a transgene encoding the pseudomature form which lacks the N-terminal 74-amino acid residue plastid-targeting sequence) in Saccharomyces cerevisiae alone does not increase taxadiene levels because of insufficient levels of the universal diterpenoid precursor geranylgeranyl diphosphate. Coexpression of Taxus chinensis taxadiene synthase and geranylgeranyl diphosphate synthase fails to increase levels. Replacement the Taxus chinensis geranylgeranyl diphosphate synthase with its counterpart from Sulfolobus acidocaldarius, which does not compete with steroid synthesis, and codon optimized the Taxus chinensis taxadiene synthase gene to ensure high-level expression, resulting in a increase in taxadiene as well as significant amounts of geranylgeraniol, suggesting taxadiene levels can be increased even further.
constitutive expression in Nicotiana benthamiana leads to de novo production of taxadiene. Transformed homozygous lines produce 11-27 microg taxadiene/g of dry weight. Treatment with an elicitor, methyl jasmonate, and metabolic pathway shunting by suppression of the phytoene synthase gene expression results in increased taxadiene accumulation by 1.4- or 1.9fold, respectively
transformation of the roots of cultured Panax ginseng C.A. Meyer to produce taxadiene. Without any change in phenotypes or growth difference, a taxadiene synthase-transgenic ginseng line accumulates 9.1 microg taxadiene per gram of dry weight. In response to the treatment of methyl jasmonate for 3 or 6 days, the accumulation is 14.6 and 15.9 microg per g of dry weight, respectively
Heterologous xpression and characterization of a "Pseudomature" form of taxadiene synthase involved in paclitaxel (taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction