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Title: New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts

Abstract

Xylose fermentation is a bottleneck in second-generation ethanol production. As such, a comprehensive understanding of xylose metabolism in naturally xylose-fermenting yeasts is essential for prospection and construction of recombinant yeast strains. The objective of the current study was to establish a reliable metabolomics protocol for quantification of key metabolites of xylose catabolism pathways in yeast, and to apply this protocol to Spathaspora arborariae. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites, and afterwards, sample preparation was optimized to examine yeast intracellular metabolites. S. arborariae was cultivated using xylose as a carbon source under aerobic and oxygen-limited conditions. Ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) were shown to efficiently quantify 14 and 5 metabolites, respectively, in a more rapid chromatographic protocol than previously described. Thirteen and eleven metabolites were quantified in S. arborariae under aerobic and oxygen-limited conditions, respectively. This targeted metabolomics protocol is shown here to quantify a total of 19 metabolites, including sugars, phosphates, coenzymes, monosaccharides, and alcohols, from xylose catabolism pathways (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) in yeast. Furthermore, to our knowledge, this is the first time that intracellular metabolites have been quantifiedmore » in S. arborariae after xylose consumption. The results indicated that fine control of oxygen levels during fermentation is necessary to optimize ethanol production by S. arborariae. The protocol presented here may be applied to other yeast species and could support yeast genetic engineering to improve second generation ethanol production. .« less

Authors:
; ; ;
Publication Date:
OSTI Identifier:
22776818
Resource Type:
Journal Article
Journal Name:
Journal of the American Society for Mass Spectrometry
Additional Journal Information:
Journal Volume: 28; Journal Issue: 12; Other Information: Copyright (c) 2017 American Society for Mass Spectrometry; http://www.springer-ny.com; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 1044-0305
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; CATABOLISM; ETHANOL; FERMENTATION; GENETIC ENGINEERING; HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY; INTERACTIONS; ION PAIRS; MASS SPECTROSCOPY; PHOSPHATES; SACCHAROSE; XYLOSE

Citation Formats

Campos, Christiane Gonçalves, Veras, Henrique César Teixeira, Aquino Ribeiro, José Antônio de, Costa, Patrícia Pinto Kalil Gonçalves, Araújo, Katiúscia Pereira, Rodrigues, Clenilson Martins, Almeida, João Ricardo Moreira de, and Abdelnur, Patrícia Verardi, E-mail: patricia.abdelnur@embrapa.br. New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts. United States: N. p., 2017. Web. doi:10.1007/S13361-017-1786-9.
Campos, Christiane Gonçalves, Veras, Henrique César Teixeira, Aquino Ribeiro, José Antônio de, Costa, Patrícia Pinto Kalil Gonçalves, Araújo, Katiúscia Pereira, Rodrigues, Clenilson Martins, Almeida, João Ricardo Moreira de, & Abdelnur, Patrícia Verardi, E-mail: patricia.abdelnur@embrapa.br. New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts. United States. doi:10.1007/S13361-017-1786-9.
Campos, Christiane Gonçalves, Veras, Henrique César Teixeira, Aquino Ribeiro, José Antônio de, Costa, Patrícia Pinto Kalil Gonçalves, Araújo, Katiúscia Pereira, Rodrigues, Clenilson Martins, Almeida, João Ricardo Moreira de, and Abdelnur, Patrícia Verardi, E-mail: patricia.abdelnur@embrapa.br. Fri . "New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts". United States. doi:10.1007/S13361-017-1786-9.
@article{osti_22776818,
title = {New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts},
author = {Campos, Christiane Gonçalves and Veras, Henrique César Teixeira and Aquino Ribeiro, José Antônio de and Costa, Patrícia Pinto Kalil Gonçalves and Araújo, Katiúscia Pereira and Rodrigues, Clenilson Martins and Almeida, João Ricardo Moreira de and Abdelnur, Patrícia Verardi, E-mail: patricia.abdelnur@embrapa.br},
abstractNote = {Xylose fermentation is a bottleneck in second-generation ethanol production. As such, a comprehensive understanding of xylose metabolism in naturally xylose-fermenting yeasts is essential for prospection and construction of recombinant yeast strains. The objective of the current study was to establish a reliable metabolomics protocol for quantification of key metabolites of xylose catabolism pathways in yeast, and to apply this protocol to Spathaspora arborariae. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites, and afterwards, sample preparation was optimized to examine yeast intracellular metabolites. S. arborariae was cultivated using xylose as a carbon source under aerobic and oxygen-limited conditions. Ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) were shown to efficiently quantify 14 and 5 metabolites, respectively, in a more rapid chromatographic protocol than previously described. Thirteen and eleven metabolites were quantified in S. arborariae under aerobic and oxygen-limited conditions, respectively. This targeted metabolomics protocol is shown here to quantify a total of 19 metabolites, including sugars, phosphates, coenzymes, monosaccharides, and alcohols, from xylose catabolism pathways (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) in yeast. Furthermore, to our knowledge, this is the first time that intracellular metabolites have been quantified in S. arborariae after xylose consumption. The results indicated that fine control of oxygen levels during fermentation is necessary to optimize ethanol production by S. arborariae. The protocol presented here may be applied to other yeast species and could support yeast genetic engineering to improve second generation ethanol production. .},
doi = {10.1007/S13361-017-1786-9},
journal = {Journal of the American Society for Mass Spectrometry},
issn = {1044-0305},
number = 12,
volume = 28,
place = {United States},
year = {2017},
month = {12}
}