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Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin

BACKGROUND: Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis...

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Autores principales: Zabala, Daniel, Song, Lijiang, Dashti, Yousef, Challis, Gregory L., Salas, José A., Méndez, Carmen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2020
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247220/
https://www.ncbi.nlm.nih.gov/pubmed/32448325
http://dx.doi.org/10.1186/s12934-020-01368-3
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author Zabala, Daniel
Song, Lijiang
Dashti, Yousef
Challis, Gregory L.
Salas, José A.
Méndez, Carmen
author_facet Zabala, Daniel
Song, Lijiang
Dashti, Yousef
Challis, Gregory L.
Salas, José A.
Méndez, Carmen
author_sort Zabala, Daniel
collection PubMed
description BACKGROUND: Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of mithramycin proceeds through the formation of the key intermediate 4-demethyl-premithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to mithramycin. However, the biosynthesis pathway for 4-demethyl-premithramycinone remains unclear. RESULTS: Expression of cosmid cosAR7, containing a set of mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-premithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-dependent acyl-CoA ligase, led to the accumulation of the tricyclic intermediate 2-hydroxy-nogalonic acid, proving its essential role in the formation of the fourth ring of 4-demethyl-premithramycinone. Expression of different sets of mithramycin biosynthesis genes as cassettes in S. albus and analysis of the resulting metabolites, allowed the reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, assigning gene functions and establishing the order of biosynthetic steps. CONCLUSIONS: We established the biosynthesis pathway for 4-demethyl-premithramycinone, and identified the minimal set of genes required for its assembly. We propose that the biosynthesis starts with the formation of a linear decaketide by the minimal polyketide synthase MtmPKS. Then, the cyclase/aromatase MtmQ catalyzes the cyclization of the first ring (C7–C12), followed by formation of the second and third rings (C5–C14; C3–C16) catalyzed by the cyclase MtmY. Formation of the fourth ring (C1–C18) requires MtmL and MtmX. Finally, further oxygenation and reduction is catalyzed by MtmOII and MtmTI/MtmTII respectively, to generate the final stable tetracyclic intermediate 4-demethyl-premithramycinone. Understanding the biosynthesis of this compound affords enhanced possibilities to generate new mithramycin analogs and improve their production titers for bioactivity investigation.
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spelling pubmed-72472202020-06-01 Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin Zabala, Daniel Song, Lijiang Dashti, Yousef Challis, Gregory L. Salas, José A. Méndez, Carmen Microb Cell Fact Research BACKGROUND: Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of mithramycin proceeds through the formation of the key intermediate 4-demethyl-premithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to mithramycin. However, the biosynthesis pathway for 4-demethyl-premithramycinone remains unclear. RESULTS: Expression of cosmid cosAR7, containing a set of mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-premithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-dependent acyl-CoA ligase, led to the accumulation of the tricyclic intermediate 2-hydroxy-nogalonic acid, proving its essential role in the formation of the fourth ring of 4-demethyl-premithramycinone. Expression of different sets of mithramycin biosynthesis genes as cassettes in S. albus and analysis of the resulting metabolites, allowed the reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, assigning gene functions and establishing the order of biosynthetic steps. CONCLUSIONS: We established the biosynthesis pathway for 4-demethyl-premithramycinone, and identified the minimal set of genes required for its assembly. We propose that the biosynthesis starts with the formation of a linear decaketide by the minimal polyketide synthase MtmPKS. Then, the cyclase/aromatase MtmQ catalyzes the cyclization of the first ring (C7–C12), followed by formation of the second and third rings (C5–C14; C3–C16) catalyzed by the cyclase MtmY. Formation of the fourth ring (C1–C18) requires MtmL and MtmX. Finally, further oxygenation and reduction is catalyzed by MtmOII and MtmTI/MtmTII respectively, to generate the final stable tetracyclic intermediate 4-demethyl-premithramycinone. Understanding the biosynthesis of this compound affords enhanced possibilities to generate new mithramycin analogs and improve their production titers for bioactivity investigation. BioMed Central 2020-05-24 /pmc/articles/PMC7247220/ /pubmed/32448325 http://dx.doi.org/10.1186/s12934-020-01368-3 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Zabala, Daniel
Song, Lijiang
Dashti, Yousef
Challis, Gregory L.
Salas, José A.
Méndez, Carmen
Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin
title Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin
title_full Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin
title_fullStr Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin
title_full_unstemmed Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin
title_short Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin
title_sort heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247220/
https://www.ncbi.nlm.nih.gov/pubmed/32448325
http://dx.doi.org/10.1186/s12934-020-01368-3
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