title: Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.
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.
authors: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark;
authors: Dan
authors: D
authors: Luo
authors: Evolva AS, CH-4153 Reinach, Switzerland;
authors: Roberta
authors: R
authors: Callari
authors: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark;
authors: Britta
authors: B
authors: Hamberger
authors: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark;
authors: Sileshi Gizachew
authors: SG
authors: Wubshet
authors: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark;
authors: Morten T
authors: MT
authors: Nielsen
authors: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark; Center for Synthetic Biology "bioSYNergy," DK-1871 Frederiksberg C, Denmark;
authors: Johan
authors: J
authors: Andersen-Ranberg
authors: Science for Life Laboratory, School of Biotechnology, Kungliga Tekniska Högskolan Royal Institute of Technology, SE-171 21 Stockholm, Sweden.
authors: Björn M
authors: BM
authors: Hallström
authors: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark;
authors: Federico
authors: F
authors: Cozzi
authors: Evolva AS, CH-4153 Reinach, Switzerland;
authors: Harald
authors: 0000-0002-3252-3119
authors: H
authors: Heider
authors: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark; Center for Synthetic Biology "bioSYNergy," DK-1871 Frederiksberg C, Denmark;
authors: Birger
authors: B
authors: Lindberg Møller
authors: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark;
authors: Dan
authors: 0000-0003-1249-1807
authors: D
authors: Staerk
authors: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark; Center for Synthetic Biology "bioSYNergy," DK-1871 Frederiksberg C, Denmark; hamberge@msu.edu.
authors: Björn
authors: B
authors: Hamberger
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000096:biosynthesis* / Q000737:chemistry; D003001:Cloning, Molecular; D003500:Cyclization; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry / Q000378:metabolism*; D028481:Euphorbia / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010666:Phenylpropionates / Q000737:chemistry / Q000378:metabolism*; D010938:Plant Oils / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome
chemical_list: C486592:3-ingenyl angelate; D000972:Antineoplastic Agents, Phytogenic; D004224:Diterpenes; D007527:Isoenzymes; D010666:Phenylpropionates; D010938:Plant Oils; D010940:Plant Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D000426:Alcohol Dehydrogenase
keywords: nonconventional cyclization; regio-specific oxidation; terpenoid biosynthesis; transcriptomic analysis