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The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data

Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techni...

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Autores principales: Tredwell, Gregory D., Edwards-Jones, Bryn, Leak, David J., Bundy, Jacob G.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025026/
https://www.ncbi.nlm.nih.gov/pubmed/21283710
http://dx.doi.org/10.1371/journal.pone.0016286
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author Tredwell, Gregory D.
Edwards-Jones, Bryn
Leak, David J.
Bundy, Jacob G.
author_facet Tredwell, Gregory D.
Edwards-Jones, Bryn
Leak, David J.
Bundy, Jacob G.
author_sort Tredwell, Gregory D.
collection PubMed
description Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris.
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spelling pubmed-30250262011-01-31 The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data Tredwell, Gregory D. Edwards-Jones, Bryn Leak, David J. Bundy, Jacob G. PLoS One Research Article Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris. Public Library of Science 2011-01-21 /pmc/articles/PMC3025026/ /pubmed/21283710 http://dx.doi.org/10.1371/journal.pone.0016286 Text en Tredwell et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Tredwell, Gregory D.
Edwards-Jones, Bryn
Leak, David J.
Bundy, Jacob G.
The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data
title The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data
title_full The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data
title_fullStr The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data
title_full_unstemmed The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data
title_short The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data
title_sort development of metabolomic sampling procedures for pichia pastoris, and baseline metabolome data
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025026/
https://www.ncbi.nlm.nih.gov/pubmed/21283710
http://dx.doi.org/10.1371/journal.pone.0016286
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