Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants
Abstract
Background: Glycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden-Meyerhof-Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism. Results: Overexpression of edd and eda in E. coli to enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP by phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this ΔpfkA mutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via the pfkA deletion significantly decreased the growth rate, while EDP overexpression in the ΔpfkA mutant failed to improve its growth rates due to metabolic burden. However, the reorganization of E. coli glycolytic strategies did reduce glucose catabolite repression. The ΔpfkA mutant in glucose medium was able to cometabolize acetate via the citric acid cycle andmore »
- Authors:
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1618650
- Alternate Identifier(s):
- OSTI ID: 1377530
- Grant/Contract Number:
- AC02-05CH11231; DESC0012722
- Resource Type:
- Published Article
- Journal Name:
- Biotechnology for Biofuels
- Additional Journal Information:
- Journal Name: Biotechnology for Biofuels Journal Volume: 9 Journal Issue: 1; Journal ID: ISSN 1754-6834
- Publisher:
- Springer Science + Business Media
- Country of Publication:
- Netherlands
- Language:
- English
- Subject:
- 09 BIOMASS FUELS; 13C; Channeling; EMP; Metabolic modeling; Synthetic biology; Catabolite repression; Xylose
Citation Formats
Hollinshead, Whitney D., Rodriguez, Sarah, Martin, Hector Garcia, Wang, George, Baidoo, Edward E. K., Sale, Kenneth L., Keasling, Jay D., Mukhopadhyay, Aindrila, and Tang, Yinjie J. Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants. Netherlands: N. p., 2016.
Web. doi:10.1186/s13068-016-0630-y.
Hollinshead, Whitney D., Rodriguez, Sarah, Martin, Hector Garcia, Wang, George, Baidoo, Edward E. K., Sale, Kenneth L., Keasling, Jay D., Mukhopadhyay, Aindrila, & Tang, Yinjie J. Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants. Netherlands. https://doi.org/10.1186/s13068-016-0630-y
Hollinshead, Whitney D., Rodriguez, Sarah, Martin, Hector Garcia, Wang, George, Baidoo, Edward E. K., Sale, Kenneth L., Keasling, Jay D., Mukhopadhyay, Aindrila, and Tang, Yinjie J. Mon .
"Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants". Netherlands. https://doi.org/10.1186/s13068-016-0630-y.
@article{osti_1618650,
title = {Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants},
author = {Hollinshead, Whitney D. and Rodriguez, Sarah and Martin, Hector Garcia and Wang, George and Baidoo, Edward E. K. and Sale, Kenneth L. and Keasling, Jay D. and Mukhopadhyay, Aindrila and Tang, Yinjie J.},
abstractNote = {Background: Glycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden-Meyerhof-Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism. Results: Overexpression of edd and eda in E. coli to enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP by phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this ΔpfkA mutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via the pfkA deletion significantly decreased the growth rate, while EDP overexpression in the ΔpfkA mutant failed to improve its growth rates due to metabolic burden. However, the reorganization of E. coli glycolytic strategies did reduce glucose catabolite repression. The ΔpfkA mutant in glucose medium was able to cometabolize acetate via the citric acid cycle and gluconeogenesis, while EDP overexpression in the ΔpfkA mutant repressed acetate flux toward gluconeogenesis. Moreover, 13C-pulse experiments in the ΔpfkA mutants showed unsequential labeling dynamics in glycolysis intermediates, possibly suggesting metabolite channeling (metabolites in glycolysis are pass from enzyme to enzyme without fully equilibrating within the cytosol medium). Conclusions: We engineered E. coli to redistribute its native glycolytic flux. The replacement of EMPP by EDP did not improve E. coli glucose utilization or biomass growth, but alleviated catabolite repression. More importantly, our results supported the hypothesis of channeling in the glycolytic pathways, a potentially overlooked mechanism for regulating glucose catabolism and coutilization of other substrates. The presence of channeling in native pathways, if proven true, would affect synthetic biology applications and metabolic modeling.},
doi = {10.1186/s13068-016-0630-y},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 9,
place = {Netherlands},
year = {Mon Oct 10 00:00:00 EDT 2016},
month = {Mon Oct 10 00:00:00 EDT 2016}
}
https://doi.org/10.1186/s13068-016-0630-y
Web of Science
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