Enzymatic synthesis of fuly stable isotope-labeled terpenoids.

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Stable isotope-labeled intermediates in terpenoid biosynthesis will be powerful probes for studies on natural terpenoids. It was previously reported that five recombinant proteins responsible for metabolism from mevalonate (MVA) to farnesyl diphosphates (FDP) were produced in E. coli, and that large scale production of [U-^<13>C_<15>] FDP from [U-^<13>C_6] MVA prepared by fermentation of an yeast was carried out. Here, enzymatic synthesis of [U-^<13>C_6] MVA and [U-^<13>C_<20>] GGDP from [U-^<13>C_2] acetate was reported.To synthesize MVA from acetate, cDNAs encoding four enzymes, acetyl-CoA synthetase (ACS), acetoacetyl-CoA synthase (AACS), HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR) were cloned based on the information of genome database of Neurospora crassa. These genes were expressed in E. coli and the recombinant proteins were produced as soluble form in E. coli. Enzyme cocktails containing four purified proteins, ATP, Coenzyme A, Mg^<2+>, NADPH and the substrate [U-^<13>C_2] acetate (99.9% ^<13>C-labeled) produced [U-^<13>C_6] MVA. GC-MS analysis of [U-^<13>C_6] MVA showed the full-scan mass spectrum without dilution of natural abundance peaks. This enzyme cocktail was combined with the other cocktails, which produce FDP and GGDP from MVA, and the combined cocktail synthesized [U-^<13>C_<15>] FDP and [U-^<13>C_<20>] GGDP from [U-^<13>C_2] acetate under the suitable reaction conditions.These fully-labeled prenyl diphosphates is powerful probes for functional analysis of gene products of terpenoid biosynthesis. We could obtain the two-dimensional C-13 NMR spectra of [U-^<13>C_<15>] FDP when ca. 1 mg of the sample was measured.