title: Enzymatic total synthesis of gibberellin A₄ from acetate.
abstract: 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. ¹³C-MVA was synthesized from ¹³C-acetate via acetyl-CoA, using four enzymes or fermentation with a MVA-secreted yeast. The diterpene hydrocarbon, ent-kaurene, was synthesized from ¹³C-acetate and ¹³C-MVA with ten and six recombinant enzymes in one test tube, respectively. Four recombinant enzymes, P450 monooxygenases and soluble dioxygenases involved in the GA₄ biosynthesis from ent-kaurene via GA₁₂ were prepared in yeast and Escherichia coli. All intermediates and the final product GA₄ were uniformly labeled with ¹³C without dilution by natural abundance when [U-¹³C₂] acetate was used. The ¹³C-NMR and MS data for [U-¹³C₂₀] ent-kaurene confirmed ¹³C-¹³C coupling, and no dilution with the ¹²C atom was observed.
authors.affiliation: United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Sugai
authors.forename: Sho
authors.initials: S
authors.lastname: Miyazaki
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Mukai
authors.forename: Isamu
authors.initials: I
authors.lastname: Yumoto
authors.forename: Masahiro
authors.initials: M
authors.lastname: Natsume
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawaide
mesh_terms: D019342:Acetic Acid / Q000378:metabolism*; D002247:Carbon Isotopes; D045786:Diterpenes, Kaurane / Q000096:biosynthesis / Q000737:chemistry; D004798:Enzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005658:Fungi / Q000201:enzymology; D005875:Gibberellins / Q000096:biosynthesis*; D010944:Plants / Q000201:enzymology; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis; D013379:Substrate Specificity
chemical_list: D002247:Carbon Isotopes; D045786:Diterpenes, Kaurane; D004798:Enzymes; D005875:Gibberellins; D011106:Polyisoprenyl Phosphates; C532593:gibberellin A4; C002627:kaurene; C002963:geranylgeranyl pyrophosphate; D019342:Acetic Acid