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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:
 [1];  [2];  [1];  [1];  [1];  [3];  [4];  [1]; ORCiD logo [2];  [3]; ORCiD logo [1];  [3]
  1. Univ. of Copenhagen (Denmark)
  2. Evolva AS, Reinach (Switzerland)
  3. Univ. of Copenhagen (Denmark); Center for Synthetic Biology, Frederiksberg (Denmark)
  4. Kungliga Tekniska Hogskolan Royal Inst. of Technology, Stockholm (Sweden)
Publication Date:
Research Org.:
Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1288983
Alternate Identifier(s):
OSTI ID: 1438201
Grant/Contract Number:  
FC02-07ER64494
Resource Type:
Journal Article: Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 113; Journal Issue: 34; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; terpenoid biosynthesis; transcriptomic analysis; regio-specific oxidation; nonconventional cyclization

Citation Formats

Luo, Dan, Callari, Roberta, Hamberger, Britta, Wubshet, Sileshi Gizachew, Nielsen, Morten T., Andersen-Ranberg, Johan, Hallstrom, Bjorn M., Cozzi, Federico, Heider, Harald, Lindberg Moller, Birger, Staerk, Dan, and Hamberger, Bjorn. Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.. United States: N. p., 2016. Web. doi:10.1073/pnas.1607504113.
Luo, Dan, Callari, Roberta, Hamberger, Britta, Wubshet, Sileshi Gizachew, Nielsen, Morten T., Andersen-Ranberg, Johan, Hallstrom, Bjorn M., Cozzi, Federico, Heider, Harald, Lindberg Moller, Birger, Staerk, Dan, & Hamberger, Bjorn. Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.. United States. doi:10.1073/pnas.1607504113.
Luo, Dan, Callari, Roberta, Hamberger, Britta, Wubshet, Sileshi Gizachew, Nielsen, Morten T., Andersen-Ranberg, Johan, Hallstrom, Bjorn M., Cozzi, Federico, Heider, Harald, Lindberg Moller, Birger, Staerk, Dan, and Hamberger, Bjorn. Tue . "Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.". United States. doi:10.1073/pnas.1607504113.
@article{osti_1288983,
title = {Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.},
author = {Luo, Dan and Callari, Roberta and Hamberger, Britta and Wubshet, Sileshi Gizachew and Nielsen, Morten T. and Andersen-Ranberg, Johan and Hallstrom, Bjorn M. and Cozzi, Federico and Heider, Harald and Lindberg Moller, Birger and Staerk, Dan and Hamberger, Bjorn},
abstractNote = {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.},
doi = {10.1073/pnas.1607504113},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 34,
volume = 113,
place = {United States},
year = {Tue Aug 09 00:00:00 EDT 2016},
month = {Tue Aug 09 00:00:00 EDT 2016}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1073/pnas.1607504113

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Cited by: 8 works
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