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:

Luo, Dan ^[1]; Callari, Roberta ^[2]; Hamberger, Britta ^[1]; Wubshet, Sileshi Gizachew ^[3]; Nielsen, Morten T. ^[1]; Andersen-Ranberg, Johan ^[4]; Hallström, Björn M. ^[5]; Cozzi, Federico ^[1]; Heider, Harald  ^[2];

• Search DOE PAGES for author "Heider, Harald"
• Search DOE PAGES for ORCID "0000-0002-3252-3119"

Lindberg Møller, Birger ^[4]; Staerk, Dan  ^[3];

• Search DOE PAGES for author "Staerk, Dan"
• Search DOE PAGES for ORCID "0000-0003-1249-1807"

Hamberger, Björn ^[4]

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+ Show Author Affiliations

1. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark,
2. Evolva AS, CH-4153 Reinach, Switzerland,
3. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark,
4. 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,
5. Science for Life Laboratory, School of Biotechnology, Kungliga Tekniska Högskolan Royal Institute of Technology, SE-171 21 Stockholm, Sweden

Publication Date:

Tue Aug 09 00:00:00 EDT 2016

Research Org.:

Univ. of Wisconsin, Madison, WI (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1288983

Alternate Identifier(s):

OSTI ID: 1438201

Grant/Contract Number:  

FC02-07ER64494

Resource Type:

Published Article

Journal Name:

Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:

Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 113 Journal Issue: 34; Journal ID: ISSN 0027-8424

Publisher:

Proceedings of the National Academy of Sciences

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; terpenoid biosynthesis; transcriptomic analysis; regio-specific oxidation; nonconventional cyclization