A cost-effective approach to produce 15N-labelled amino acids employing Chlamydomonas reinhardtii CC503

Microbial Cell Factories

Technical Notes

María Jesús Cañal¹  Luis Valledor¹  Juan Manuel Marchante-Gayón²  José Ignacio García Alonso²  Pablo Rodríguez González²  Jesús Nicolás Carcelén² 

[1] Department of Organisms and Systems Biology, University of Oviedo, C/Catedrático Rodrigo Uría s/n, 33071, Oviedo, Spain;Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006, Oviedo, Spain;

关键词: Biosynthesis;    Chlamydomonas reinhardtii;    N-labelled amino acids;    Isotopic enrichment;    Protein extraction;    GC–MS;   

DOI  :  10.1186/s12934-017-0759-9

 received in 2017-05-25, accepted in 2017-08-10,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsThe aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15NH4Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15N-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.ConclusionsPreviously 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 13C-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 15N-labelled amino acids could be obtained.

【 授权许可】

CC BY   
© The Author(s) 2017