Enzymatic Total Synthesis of Gibberellin A4 from Acetate

• Published in: Bioscience, biotechnology, and biochemistry Vol. 75; no. 1; pp. 128 - 135
• Main Authors: SUGAI, Yoshinori, MIYAZAKI, Sho, MUKAI, Shinichiro, YUMOTO, Isamu, NATSUME, Masahiro, KAWAIDE, Hiroshi
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 2011; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Acetic Acid - metabolism; Bioconversions. Hemisynthesis; Biological and medical sciences; biosynthesis; Biotechnology; C-NMR; Carbon Isotopes; Diterpenes, Kaurane - biosynthesis; Diterpenes, Kaurane - chemistry; Enzymes - biosynthesis; Enzymes - genetics; Enzymes - metabolism; Fundamental and applied biological sciences. Psychology; Fungi - enzymology; Gibberellins - biosynthesis; isotope; Methods. Procedures. Technologies; Plants - enzymology; Polyisoprenyl Phosphates - biosynthesis; recombinant enzyme; Substrate Specificity; terpenoid; Enzymatic synthesis; Terpenoid; Enzyme; Gibberellin; Biosynthesis; NMR spectrometry; Acetate; isotope; C-NMR; Isotopes; recombinant enzyme
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.100733

Natural terpenoids have elaborate structures and various bioactivities, making difficult their synthesis and labeling with isotopes. We report here the enzymatic total synthesis of plant hormone gibberellins (GAs) with recombinant biosynthetic enzymes from stable isotope-labeled acetate. Mevalonate (MVA) is a key intermediate for the terpenoid biosynthetic pathway. 13 C-MVA was synthesized from 13 C-acetate via acetyl-CoA, using four enzymes or fermentation with a MVA-secreted yeast. The diterpene hydrocarbon, ent-kaurene, was synthesized from 13 C-acetate and 13 C-MVA with ten and six recombinant enzymes in one test tube, respectively. Four recombinant enzymes, P450 monooxygenases and soluble dioxygenases involved in the GA 4 biosynthesis from ent-kaurene via GA 12 were prepared in yeast and Escherichia coli. All intermediates and the final product GA 4 were uniformly labeled with 13 C without dilution by natural abundance when [U- 13 C 2 ] acetate was used. The 13 C-NMR and MS data for [U- 13 C 20 ] ent-kaurene confirmed 13 C- 13 C coupling, and no dilution with the 12 C atom was observed.