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A cost-effective approach to produce (15)N-labelled amino acids employing Chlamydomonas reinhardtii CC503
BACKGROUND: The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the interna...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563056/ https://www.ncbi.nlm.nih.gov/pubmed/28821247 http://dx.doi.org/10.1186/s12934-017-0759-9 |
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author | Nicolás Carcelén, Jesús Marchante-Gayón, Juan Manuel González, Pablo Rodríguez Valledor, Luis Cañal, María Jesús Alonso, José Ignacio García |
author_facet | Nicolás Carcelén, Jesús Marchante-Gayón, Juan Manuel González, Pablo Rodríguez Valledor, Luis Cañal, María Jesús Alonso, José Ignacio García |
author_sort | Nicolás Carcelén, Jesús |
collection | PubMed |
description | BACKGROUND: The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. RESULTS: The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, (15)N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing (15)NH(4)Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the (15)N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC–MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. CONCLUSIONS: Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only (13)C-labelled fatty acids have been isolated from labelled microalga biomass as valuable industrial products. In this study, we propose Chlamydomonas reinhardtii CC503 as a feasible microorganism and strain to produce labelled biomass from which a standard containing sixteen (15)N-labelled amino acids could be obtained. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-017-0759-9) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5563056 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-55630562017-08-21 A cost-effective approach to produce (15)N-labelled amino acids employing Chlamydomonas reinhardtii CC503 Nicolás Carcelén, Jesús Marchante-Gayón, Juan Manuel González, Pablo Rodríguez Valledor, Luis Cañal, María Jesús Alonso, José Ignacio García Microb Cell Fact Technical Notes BACKGROUND: The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. RESULTS: The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, (15)N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing (15)NH(4)Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the (15)N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC–MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. CONCLUSIONS: Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only (13)C-labelled fatty acids have been isolated from labelled microalga biomass as valuable industrial products. In this study, we propose Chlamydomonas reinhardtii CC503 as a feasible microorganism and strain to produce labelled biomass from which a standard containing sixteen (15)N-labelled amino acids could be obtained. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-017-0759-9) contains supplementary material, which is available to authorized users. BioMed Central 2017-08-18 /pmc/articles/PMC5563056/ /pubmed/28821247 http://dx.doi.org/10.1186/s12934-017-0759-9 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. |
spellingShingle | Technical Notes Nicolás Carcelén, Jesús Marchante-Gayón, Juan Manuel González, Pablo Rodríguez Valledor, Luis Cañal, María Jesús Alonso, José Ignacio García A cost-effective approach to produce (15)N-labelled amino acids employing Chlamydomonas reinhardtii CC503 |
title | A cost-effective approach to produce (15)N-labelled amino acids employing Chlamydomonas reinhardtii CC503 |
title_full | A cost-effective approach to produce (15)N-labelled amino acids employing Chlamydomonas reinhardtii CC503 |
title_fullStr | A cost-effective approach to produce (15)N-labelled amino acids employing Chlamydomonas reinhardtii CC503 |
title_full_unstemmed | A cost-effective approach to produce (15)N-labelled amino acids employing Chlamydomonas reinhardtii CC503 |
title_short | A cost-effective approach to produce (15)N-labelled amino acids employing Chlamydomonas reinhardtii CC503 |
title_sort | cost-effective approach to produce (15)n-labelled amino acids employing chlamydomonas reinhardtii cc503 |
topic | Technical Notes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563056/ https://www.ncbi.nlm.nih.gov/pubmed/28821247 http://dx.doi.org/10.1186/s12934-017-0759-9 |
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