15.^<31>P-NMRイメージングによる酵素合成系の改良と酢酸を原料にしたジベレリンA_4の酵素的全合成(口頭発表)

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Terpenoids are widely distributed in nature and contain many bioactive secondary metabolites. The enzymatic total synthesis and isotope labeling of natural terpenoids will be powerful probes for studying the biosynthesis of terpenoids. Here, we successfully synthesized a bioactive giberrellin from acetate with biosynthetic enzymes. When [U-^<13>C_2]acetate was used for the starting material, [U-^<13>C_<20>]ent-kaurene was enzymatically synthesized via mevalonate. The ^<13>C-NMR of [U-^<13>C_<20>]ent-kaurene observed signals to assign all peaks from chemical shifts values and ^<13>C-^<13>C coupling constants. GA_4-biosynthetic genes encoding two P450 monooxygenases and two dioxygenases were expressed in E. coli or Pichia yeast, respectively. [U-^<13>C_<20>]GA_<12> was synthesized from [U-^<13>C_<20>]ent-kaurene containing two P450 monooxygenases. Finally, addition of two soluble dioxygenases into the [U-^<13>C_<20>]GA_<12> synthetic cocktail led to synthesis of [U-^<13>C_<19>]GA_4. In addition, we will report also ^<31>P-NMR monitoring of intermediates during enzyme reactions from mevalonate to isopentenyl diphosphate. We optimized the enzyme reaction condition by ^<31>P-NMR technique.
2010-10-01