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Title: Metabolomics Revealed an Association of Metabolite Changes and Defective Growth in Methylobacterium extorquens AM1 Overexpressing ecm during Growth on Methanol

Abstract

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multi-carbon compounds. The ethylmalonyl-CoA (EMC) pathway is one of the central assimilatory pathways in M. extorquens during growth on C1 and C2 substrates. Previous studies had shown that ethylmalonyl-CoA mutase functioned as a control point during the transition from growth on succinate to growth on ethylamine. In this study we overexpressed ecm, phaA, mcmAB and found that upregulating ecm by expressing it from the strong constitutive mxaF promoter caused a 27% decrease in growth rate on methanol compared to the strain with an empty vector. Targeted metabolomics demonstrated that most of the central intermediates in the ecm over-expressing strain did not change significantly compared to the control strain; However, poly-β-hydroxybutyrate (PHB) was 4.5-fold lower and 3-hydroxybutyryl-CoA was 1.6-fold higher. Moreover, glyoxylate, a toxic and highly regulated essential intermediate, was determined to be 2.6-fold higher when ecm was overexpressed. These results demonstrated that overexpressing ecm can manipulate carbon flux through the EMC pathway and divert it from the carbon and energy storage product PHB, leading to an accumulation of glyoxylate. Furthermore, untargeted metabolomics discovered two unusual metabolites, alanine (Ala)-meso-diaminopimelic acid (mDAP) and Ala-mDAP-Ala, each over 45-fold highermore » in the ecm overexpressing strain. These two peptides were also found to be highly produced in a dose-dependent manner when glyoxylate was added to the control strain. Overall, this work has explained a direct association of ecm overexpression with glyoxylate accumulation up to a toxic level, which inhibits cell growth on methanol. Lastly, this research provides useful insight for manipulating the EMC pathway for efficiently producing high-value chemicals in M. extorquens.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
Univ. of Washington, Seattle, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1346604
Alternate Identifier(s):
OSTI ID: 1257762
Grant/Contract Number:  
DESC0006871; SC0006871
Resource Type:
Published Article
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Name: PLoS ONE Journal Volume: 11 Journal Issue: 4; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science (PLoS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; metabolomics; serine; metabolites; liquid chromatography-mass spectrometry; enzyme metabolism; database searching; metabolic pathways; protein metabolism

Citation Formats

Cui, Jinyu, Good, Nathan M., Hu, Bo, Yang, Jing, Wang, Qianwen, Sadilek, Martin, Yang, Song, and Berg, ed., Ivan A. Metabolomics Revealed an Association of Metabolite Changes and Defective Growth in Methylobacterium extorquens AM1 Overexpressing ecm during Growth on Methanol. United States: N. p., 2016. Web. doi:10.1371/journal.pone.0154043.
Cui, Jinyu, Good, Nathan M., Hu, Bo, Yang, Jing, Wang, Qianwen, Sadilek, Martin, Yang, Song, & Berg, ed., Ivan A. Metabolomics Revealed an Association of Metabolite Changes and Defective Growth in Methylobacterium extorquens AM1 Overexpressing ecm during Growth on Methanol. United States. doi:10.1371/journal.pone.0154043.
Cui, Jinyu, Good, Nathan M., Hu, Bo, Yang, Jing, Wang, Qianwen, Sadilek, Martin, Yang, Song, and Berg, ed., Ivan A. Tue . "Metabolomics Revealed an Association of Metabolite Changes and Defective Growth in Methylobacterium extorquens AM1 Overexpressing ecm during Growth on Methanol". United States. doi:10.1371/journal.pone.0154043.
@article{osti_1346604,
title = {Metabolomics Revealed an Association of Metabolite Changes and Defective Growth in Methylobacterium extorquens AM1 Overexpressing ecm during Growth on Methanol},
author = {Cui, Jinyu and Good, Nathan M. and Hu, Bo and Yang, Jing and Wang, Qianwen and Sadilek, Martin and Yang, Song and Berg, ed., Ivan A.},
abstractNote = {Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multi-carbon compounds. The ethylmalonyl-CoA (EMC) pathway is one of the central assimilatory pathways in M. extorquens during growth on C1 and C2 substrates. Previous studies had shown that ethylmalonyl-CoA mutase functioned as a control point during the transition from growth on succinate to growth on ethylamine. In this study we overexpressed ecm, phaA, mcmAB and found that upregulating ecm by expressing it from the strong constitutive mxaF promoter caused a 27% decrease in growth rate on methanol compared to the strain with an empty vector. Targeted metabolomics demonstrated that most of the central intermediates in the ecm over-expressing strain did not change significantly compared to the control strain; However, poly-β-hydroxybutyrate (PHB) was 4.5-fold lower and 3-hydroxybutyryl-CoA was 1.6-fold higher. Moreover, glyoxylate, a toxic and highly regulated essential intermediate, was determined to be 2.6-fold higher when ecm was overexpressed. These results demonstrated that overexpressing ecm can manipulate carbon flux through the EMC pathway and divert it from the carbon and energy storage product PHB, leading to an accumulation of glyoxylate. Furthermore, untargeted metabolomics discovered two unusual metabolites, alanine (Ala)-meso-diaminopimelic acid (mDAP) and Ala-mDAP-Ala, each over 45-fold higher in the ecm overexpressing strain. These two peptides were also found to be highly produced in a dose-dependent manner when glyoxylate was added to the control strain. Overall, this work has explained a direct association of ecm overexpression with glyoxylate accumulation up to a toxic level, which inhibits cell growth on methanol. Lastly, this research provides useful insight for manipulating the EMC pathway for efficiently producing high-value chemicals in M. extorquens.},
doi = {10.1371/journal.pone.0154043},
journal = {PLoS ONE},
number = 4,
volume = 11,
place = {United States},
year = {2016},
month = {4}
}

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DOI: 10.1371/journal.pone.0154043

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