60.酢酸を出発原料としたゲラニルゲラニル二リン酸の酵素的合成(口頭発表)

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Stable isotope-labeled intermediates in isoprenoid biosynthesis will be powerful probes for studies on natural isoprenoids. We 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 succeeded. Here, we report enzymatic synthesis of [U-^^<13>C_6] MVA and [U-^^<13>C_<20>] GGDP from [U-^^<13>C_2] acetate. To synthesize MVA from acetate, four enzymes are involved; acetyl-CoA synthetase (ACS), acetoacetyl-CoA synthase (AACS), HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR). cDNAs encoding these enzymes were cloned based on the information of genome database of Neurospora crassa. It was reported that HMGR is catalytically active without membrane anchor domain.For this reason, soluble HMGR was prepared by truncation of N-terminal 664 amino acid residues. Each open reading frame of these cDNAs was ligated into pQE-30 vector to produce the recombinant proteins 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 MVA. GC-MS analysis of [U-^^<13>C_6] MVA from [U-^^<13>C_2] acetate showed the spectrum without dilution of a natural abundance peaks. Furthermore, [U-^^<13>C_<20>] GGDP was successfully synthesized in addition of six enzymes for metabolism of GGDP from MVA into the above enzyme cocktail.
植物化学調節学会の論文
2007-10-05

60.酢酸を出発原料としたゲラニルゲラニル二リン酸の酵素的合成(口頭発表)

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