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. 2016 Aug 23;113(34):E5082-9.
doi: 10.1073/pnas.1607504113. Epub 2016 Aug 9.

Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L

Dan Luo  1 , Roberta Callari  2 , Britta Hamberger  1 , Sileshi Gizachew Wubshet  3 , Morten T Nielsen  1 , Johan Andersen-Ranberg  4 , Björn M Hallström  5 , Federico Cozzi  1 , Harald Heider  2 , Birger Lindberg Møller  4 , Dan Staerk  3 , Björn Hamberger  6
Affiliations

Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L

Dan Luo et al. Proc Natl Acad Sci U S A. .

Abstract

The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon-carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids.

Keywords: nonconventional cyclization; regio-specific oxidation; terpenoid biosynthesis; transcriptomic analysis.

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Conflict of interest statement

D.L., M.T.N., R.C., and Björn Hamberger have filed a patent application (PA 2015 70069) covering “Methods and materials for producing oxidized macrocyclic diterpenes” related to the manufacture of jolkinol C and precursors thereof.

Figures

Fig. 1.
Fig. 1.
Distribution and structures of bioactive Euphorbia diterpenes and their carbon skeletons.
Fig. 2.
Fig. 2.
Functional characterization of E. lathyris CBS, P450s, and ADH1 via in vivo expression. (A) GC-MS profiles of N. benthamiana transiently expressing E. lathyris CBS, internal standard: fluoranthene, 1 ppm. (B) LC-HRMS total ion chromatograms of N. benthamiana transiently expressing E. lathyris CBS, CYP71D445, CYP726A27, and ADH1. (C) Structures of isolated intermediates and products. (D) LC-HRMS total ion chromatograms of S. cerevisiae expressing CBS, CYP71D445, CYP726A27, and ADH1.
Fig. 3.
Fig. 3.
Investigation of in vitro assayed CYP71D445, CYP726A27, and ADH1 by LC-HRMS analysis. (A) LC-HRMS total ion chromatograms of the products generated from recombinant ADH1 when supplied with 3, 4, 6, 7, and 8. (B) LC-HRMS total ion chromatograms of the products generated by combined assays of microsomal fractions expressing CYP71D445, CYP726A27, and the recombinant ADH1, supplied with casbene.
Fig. 4.
Fig. 4.
Characterization of catalytic features of CYP71D445 and CYP726A27 by combined in vitro assays. (A) LC-HRMS total ion chromatograms of the products generated by combined assays of microsomes expressing CYP71D445, CYP726A27, and the recombinant ADH1 supplied with 6. (B) LC-HRMS total ion chromatograms of the products generated by combined assays of microsomes expressing CYP71D445, CYP726A27, and the recombinant ADH1 supplied with 3.
Fig. 5.
Fig. 5.
Proposed pathway for the conversion of casbene (1) to jolkinol C (5) through enzymatic reactions catalyzed by CYP71D445, CYP726A27, and E. lathyris ADH1.
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