gas-phase and enzyme-free post-transition state direct dynamics simulation gas-phase reveal that abietadiene synthase must intervene in order to produce abietadiene selectively, steering this reaction to avoid the generation of a sevenmembered ring containing product which is formed in the enzyme-free reaction
initial product of the reaction, thermally unstable. Dehydration of the alcohol products of (+)-copalyl diphosphate, yielding the well established diterpene products levopimaradiene, abietadiene, neoabietadiene, and palustradiene, may occurr due to three conditions of the GC-MS analysis typically used for identification of diterpene synthase products: a hot injector, a hot oven temperature necessary for eluting the compounds, and the high temperature and high energy of the MS and its interface
resin duct epithelial cells are the main site of diterpene biosynthesis in Sitka spruce, diterpenoid biosynthesis is induced in cortical resin duct epithelial cells early upon treatment with methyljasmonate, and immature developing traumatic resin duct epithelial cells produce levopimaradiene/abietadiene synthase
Site-directed mutagenesis is carried out via PCR amplification with overlapping mutagenic primers, and the mutant genes verify by complete sequencing. The resulting wild type and mutant genes are then transferred via directional recombination to the T7-promoter and N-terminal 6his fusion expression vector pDEST17. Use of the pDEST17 vector results in a 25 amino acid residue linker between the 6 His-tag and the cloned protein.
swapping of residues 568640 of isopimaradiene synhase to corresponding residues 560-632 of levopimaradiene/abietadiene synthase results in complete reversion of the product profiles of the two enzymes
increase of levopimaradiene synthesis in Escherichia coli by amplification of the flux toward isopentenyl diphosphate and dimethylallyl diphosphate precursors and reprogramming the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase and levopimaradiene synthase. The most productive pathway, combining precursor flux amplification and mutant synthases, confers approximately 2600fold increase in levopimaradiene levels. A maximum titer of approximately 700 mg/l is obtained by cultivation in a benchscale bioreactor