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Title: Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization

Abstract

Ethylene glycol is used as a raw material in the production of polyethylene terephthalate, in antifreeze, as a gas hydrate inhibitor in pipelines, and for many other industrial applications. It is metabolized by aerobic microbial processes via the highly toxic intermediates glycolaldehyde and glycolate through C2 metabolic pathways. Pseudomonas putida KT2440, which has been engineered for environmental remediation applications given its high toxicity tolerance and broad substrate specificity, is not able to efficiently metabolize ethylene glycol, despite harboring putative genes for this purpose. To further expand the metabolic portfolio of P. putida, we elucidated the metabolic pathway to enable ethylene glycol via systematic overexpression of glyoxylate carboligase (gcl) in combination with other genes. Quantitative reverse transcription polymerase chain reaction demonstrated that all of the four genes in genomic proximity to gcl (hyi, glxR, ttuD, and pykF) are transcribed as an operon. Where the expression of only two genes (gcl and glxR) resulted in growth in ethylene glycol, improved growth and ethylene glycol utilization were observed when the entire gcl operon was expressed. Both glycolaldehyde and glyoxal inhibit growth in concentrations of ethylene glycol above 50 mM. To overcome this bottleneck, the additional overexpression of the glycolate oxidase (glcDEF) operon removesmore » the glycolate bottleneck and minimizes the production of these toxic intermediates, permitting growth in up to 2 M (~124 g/L) and complete consumption of 0.5 M (31 g/L) ethylene glycol in shake flask experiments. In addition, the engineered strain enables conversion of ethylene glycol to medium-chain-length polyhydroxyalkanoates (mcl-PHAs). Overall, this study provides a robust P. putida KT2440 strain for ethylene glycol consumption, which will serve as a foundational strain for further biocatalyst development for applications in the remediation of waste polyester plastics and biomass-derived wastewater streams.« less

Authors:
; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
OSTI Identifier:
1457660
Report Number(s):
NREL/JA-2A00-71826
Journal ID: ISSN 1096-7176
DOE Contract Number:
AC36-08GO28308
Resource Type:
Journal Article
Resource Relation:
Journal Name: Metabolic Engineering; Journal Volume: 48; Journal Issue: C
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; ethylene glycol; Pseudomonas putida kt2440 glyoxylate; glycolate; glycolaldehyde; metabolism

Citation Formats

Franden, Mary Ann, Jayakody, Lahiru N., Li, Wing-Jin, Wagner, Neil J., Cleveland, Nicholas S., Michener, William E., Hauer, Bernhard, Blank, Lars M., Wierckx, Nick, Klebensberger, Janosch, and Beckham, Gregg T.. Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization. United States: N. p., 2018. Web. doi:10.1016/j.ymben.2018.06.003.
Franden, Mary Ann, Jayakody, Lahiru N., Li, Wing-Jin, Wagner, Neil J., Cleveland, Nicholas S., Michener, William E., Hauer, Bernhard, Blank, Lars M., Wierckx, Nick, Klebensberger, Janosch, & Beckham, Gregg T.. Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization. United States. doi:10.1016/j.ymben.2018.06.003.
Franden, Mary Ann, Jayakody, Lahiru N., Li, Wing-Jin, Wagner, Neil J., Cleveland, Nicholas S., Michener, William E., Hauer, Bernhard, Blank, Lars M., Wierckx, Nick, Klebensberger, Janosch, and Beckham, Gregg T.. Sun . "Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization". United States. doi:10.1016/j.ymben.2018.06.003.
@article{osti_1457660,
title = {Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization},
author = {Franden, Mary Ann and Jayakody, Lahiru N. and Li, Wing-Jin and Wagner, Neil J. and Cleveland, Nicholas S. and Michener, William E. and Hauer, Bernhard and Blank, Lars M. and Wierckx, Nick and Klebensberger, Janosch and Beckham, Gregg T.},
abstractNote = {Ethylene glycol is used as a raw material in the production of polyethylene terephthalate, in antifreeze, as a gas hydrate inhibitor in pipelines, and for many other industrial applications. It is metabolized by aerobic microbial processes via the highly toxic intermediates glycolaldehyde and glycolate through C2 metabolic pathways. Pseudomonas putida KT2440, which has been engineered for environmental remediation applications given its high toxicity tolerance and broad substrate specificity, is not able to efficiently metabolize ethylene glycol, despite harboring putative genes for this purpose. To further expand the metabolic portfolio of P. putida, we elucidated the metabolic pathway to enable ethylene glycol via systematic overexpression of glyoxylate carboligase (gcl) in combination with other genes. Quantitative reverse transcription polymerase chain reaction demonstrated that all of the four genes in genomic proximity to gcl (hyi, glxR, ttuD, and pykF) are transcribed as an operon. Where the expression of only two genes (gcl and glxR) resulted in growth in ethylene glycol, improved growth and ethylene glycol utilization were observed when the entire gcl operon was expressed. Both glycolaldehyde and glyoxal inhibit growth in concentrations of ethylene glycol above 50 mM. To overcome this bottleneck, the additional overexpression of the glycolate oxidase (glcDEF) operon removes the glycolate bottleneck and minimizes the production of these toxic intermediates, permitting growth in up to 2 M (~124 g/L) and complete consumption of 0.5 M (31 g/L) ethylene glycol in shake flask experiments. In addition, the engineered strain enables conversion of ethylene glycol to medium-chain-length polyhydroxyalkanoates (mcl-PHAs). Overall, this study provides a robust P. putida KT2440 strain for ethylene glycol consumption, which will serve as a foundational strain for further biocatalyst development for applications in the remediation of waste polyester plastics and biomass-derived wastewater streams.},
doi = {10.1016/j.ymben.2018.06.003},
journal = {Metabolic Engineering},
number = C,
volume = 48,
place = {United States},
year = {Sun Jul 01 00:00:00 EDT 2018},
month = {Sun Jul 01 00:00:00 EDT 2018}
}