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Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform
Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
National Academy of Sciences
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9894180/ https://www.ncbi.nlm.nih.gov/pubmed/36442128 http://dx.doi.org/10.1073/pnas.2215372119 |
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author | Chavez, Benjamin G. Srinivasan, Prashanth Glockzin, Kayla Kim, Neill Montero Estrada, Olga Jirschitzka, Jan Rowden, Gage Shao, Jonathan Meinhardt, Lyndel Smolke, Christina D. D’Auria, John C. |
author_facet | Chavez, Benjamin G. Srinivasan, Prashanth Glockzin, Kayla Kim, Neill Montero Estrada, Olga Jirschitzka, Jan Rowden, Gage Shao, Jonathan Meinhardt, Lyndel Smolke, Christina D. D’Auria, John C. |
author_sort | Chavez, Benjamin G. |
collection | PubMed |
description | Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway. |
format | Online Article Text |
id | pubmed-9894180 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | National Academy of Sciences |
record_format | MEDLINE/PubMed |
spelling | pubmed-98941802023-05-28 Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform Chavez, Benjamin G. Srinivasan, Prashanth Glockzin, Kayla Kim, Neill Montero Estrada, Olga Jirschitzka, Jan Rowden, Gage Shao, Jonathan Meinhardt, Lyndel Smolke, Christina D. D’Auria, John C. Proc Natl Acad Sci U S A Biological Sciences Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway. National Academy of Sciences 2022-11-28 2022-12-06 /pmc/articles/PMC9894180/ /pubmed/36442128 http://dx.doi.org/10.1073/pnas.2215372119 Text en Copyright © 2022 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) . |
spellingShingle | Biological Sciences Chavez, Benjamin G. Srinivasan, Prashanth Glockzin, Kayla Kim, Neill Montero Estrada, Olga Jirschitzka, Jan Rowden, Gage Shao, Jonathan Meinhardt, Lyndel Smolke, Christina D. D’Auria, John C. Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform |
title | Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform |
title_full | Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform |
title_fullStr | Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform |
title_full_unstemmed | Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform |
title_short | Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform |
title_sort | elucidation of tropane alkaloid biosynthesis in erythroxylum coca using a microbial pathway discovery platform |
topic | Biological Sciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9894180/ https://www.ncbi.nlm.nih.gov/pubmed/36442128 http://dx.doi.org/10.1073/pnas.2215372119 |
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