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Title: Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph

Abstract

Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene (pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determined by 13C-labeling in intracellular metabolites. As a result, introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.

Authors:
 [1];  [1];  [1];  [1];  [1]
  1. Univ. of Delaware, Newark, DE (United States)
Publication Date:
Research Org.:
Univ. of Delaware, Newark, DE (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
OSTI Identifier:
1411402
Alternate Identifier(s):
OSTI ID: 1703839
Grant/Contract Number:  
AR0000432
Resource Type:
Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 45; Journal Issue: C; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; Synthetic methylotrophy; Methanol; Escherichia coli; Pentose phosphate pathway; Phosphoglucose isomerase

Citation Formats

Bennett, R. Kyle, Gonzalez, Jacqueline E., Whitaker, W. Brian, Antoniewicz, Maciek R., and Papoutsakis, Eleftherios T. Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph. United States: N. p., 2017. Web. doi:10.1016/j.ymben.2017.11.016.
Bennett, R. Kyle, Gonzalez, Jacqueline E., Whitaker, W. Brian, Antoniewicz, Maciek R., & Papoutsakis, Eleftherios T. Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph. United States. https://doi.org/10.1016/j.ymben.2017.11.016
Bennett, R. Kyle, Gonzalez, Jacqueline E., Whitaker, W. Brian, Antoniewicz, Maciek R., and Papoutsakis, Eleftherios T. Tue . "Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph". United States. https://doi.org/10.1016/j.ymben.2017.11.016. https://www.osti.gov/servlets/purl/1411402.
@article{osti_1411402,
title = {Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph},
author = {Bennett, R. Kyle and Gonzalez, Jacqueline E. and Whitaker, W. Brian and Antoniewicz, Maciek R. and Papoutsakis, Eleftherios T.},
abstractNote = {Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene (pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determined by 13C-labeling in intracellular metabolites. As a result, introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.},
doi = {10.1016/j.ymben.2017.11.016},
journal = {Metabolic Engineering},
number = C,
volume = 45,
place = {United States},
year = {2017},
month = {12}
}

Journal Article:

Citation Metrics:
Cited by: 13 works
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Figures / Tables:

Figure 1 Figure 1: Strategies to enhance synthetic methylotrophy. (A): Methanol is oxidized by the mdh-encoded MDH to formaldehyde, which is assimilated via the ribulose monophosphate (RuMP) pathway (hps, phi) to F6P upon reacting with Ru5P (green). Deletion of frmA eliminates formaldehyde dissimilation to CO2 to conserve methanol carbon. The heterologous non-oxidativemore » pentose phosphate pathway (PPP) from Bacillus methanolicus (rpe, fba, glpX, pfk, tkt) generates Ru5P from F6P (blue). Deletion of pgi reroutes glucose catabolism through the oxidative PPP to generate Ru5P from G6P. (B, C): Glucose carbon flux in methylotrophic E. coli ΔfrmA (B) and methylotrophic E. coli ΔfrmAΔpgi (C). Enzymes (genes): methanol dehydrogenase (mdh), hexulose phosphate synthase (hps), phosphohexulose isomerase (phi), phosphoglucose isomerase (pgi), formaldehyde dehydrogenase (frmA), phosphofructokinase (pfk), fructose-bisphosphate aldolase (fba), transketolase (tkt), ribulose phosphate epimerase (rpe), sedoheptulose bisphosphate (glpX), fructose bisphosphatase (fbp), ribose phosphate isomerase (rpi). Metabolites: glucose 6-phosphate (G6P), 6-phosphogluconolactone (GL6P), 6-phosphogluconate (6PG), ribulose 5-phosphate (Ru5P), ribose 5-phosphate (R5P), hexulose 6-phosphate (H6P), fructose 6-phosphate (F6P), fructose bisphosphate (FBP), dihydroxyacetone phosphate (DHAP), glyceraldehyde 3-phosphate (GAP), xylulose 5-phosphate (X5P), erythrose 4-phosphate (E4P), sedoheptulose bisphosphate (SBP), sedoheptulose 7-phosphate (S7P).« less

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Works referenced in this record:

Parallel labeling experiments for pathway elucidation and 13C metabolic flux analysis
journal, December 2015


Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements
journal, July 2006

  • Antoniewicz, Maciek R.; Kelleher, Joanne K.; Stephanopoulos, Gregory
  • Metabolic Engineering, Vol. 8, Issue 4
  • DOI: 10.1016/j.ymben.2006.01.004

Elementary metabolite units (EMU): A novel framework for modeling isotopic distributions
journal, January 2007

  • Antoniewicz, Maciek R.; Kelleher, Joanne K.; Stephanopoulos, Gregory
  • Metabolic Engineering, Vol. 9, Issue 1
  • DOI: 10.1016/j.ymben.2006.09.001

Measuring Deuterium Enrichment of Glucose Hydrogen Atoms by Gas Chromatography/Mass Spectrometry
journal, April 2011

  • Antoniewicz, Maciek R.; Kelleher, Joanne K.; Stephanopoulos, Gregory
  • Analytical Chemistry, Vol. 83, Issue 8
  • DOI: 10.1021/ac200012p

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants the Keio collection
journal, February 2006

  • Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki
  • Molecular Systems Biology, Vol. 2, Article No. 2006.0008
  • DOI: 10.1038/msb4100050

Overflow metabolism in Escherichia coli results from efficient proteome allocation
journal, December 2015

  • Basan, Markus; Hui, Sheng; Okano, Hiroyuki
  • Nature, Vol. 528, Issue 7580
  • DOI: 10.1038/nature15765

Expression of Clostridium acetobutylicumATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification
journal, March 1998

  • Bermejo, Lourdes L.; Welker, Neil E.; Papoutsakis, Eleftherios T.
  • Applied and Environmental Microbiology, Vol. 64, Issue 3
  • DOI: 10.1128/AEM.64.3.1079-1085.1998

Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites
journal, November 2013

  • Espah Borujeni, Amin; Channarasappa, Anirudh S.; Salis, Howard M.
  • Nucleic Acids Research, Vol. 42, Issue 4
  • DOI: 10.1093/nar/gkt1139

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus
journal, February 2004


Genetic Basis of Growth Adaptation of Escherichia coli after Deletion of pgi, a Major Metabolic Gene
journal, November 2010


Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. I. Fast transients induced by methanol pulses and methanol accumulation
journal, January 1987

  • Chu, I-Ming; Papoutsakis, Eleftherios Terry
  • Biotechnology and Bioengineering, Vol. 29, Issue 1, p. 55-64
  • DOI: 10.1002/bit.260290109

Integrated 13 C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli
journal, March 2015


Optimal tracers for parallel labeling experiments and 13C metabolic flux analysis: A new precision and synergy scoring system
journal, November 2016


Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae
journal, December 2017


One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
journal, May 2000

  • Datsenko, K. A.; Wanner, B. L.
  • Proceedings of the National Academy of Sciences, Vol. 97, Issue 12, p. 6640-6645
  • DOI: 10.1073/pnas.120163297

Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli
journal, January 2017

  • Englaender, Jacob A.; Jones, J. Andrew; Cress, Brady F.
  • ACS Synthetic Biology, Vol. 6, Issue 4
  • DOI: 10.1021/acssynbio.6b00350

Correction of13C Mass Isotopomer Distributions for Natural Stable Isotope Abundance
journal, March 1996


Identification and validation of reference genes to study the gene expression in Gluconacetobacter diazotrophicus grown in different carbon sources using RT-qPCR
journal, October 2012

  • Galisa, Péricles S.; da Silva, Helder A. P.; Macedo, Aline V. M.
  • Journal of Microbiological Methods, Vol. 91, Issue 1
  • DOI: 10.1016/j.mimet.2012.07.005

Molecular Basis of Formaldehyde Detoxification: CHARACTERIZATION OF TWO S-FORMYLGLUTATHIONE HYDROLASES FROM ESCHERICHIA COLI, FrmB AND YeiG
journal, March 2006

  • Gonzalez, Claudio F.; Proudfoot, Michael; Brown, Greg
  • Journal of Biological Chemistry, Vol. 281, Issue 20
  • DOI: 10.1074/jbc.M600996200

Tracing metabolism from lignocellulosic biomass and gaseous substrates to products with stable-isotopes
journal, February 2017


Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis
journal, January 2017

  • Gonzalez, Jacqueline E.; Long, Christopher P.; Antoniewicz, Maciek R.
  • Metabolic Engineering, Vol. 39
  • DOI: 10.1016/j.ymben.2016.11.003

Rethinking biological activation of methane and conversion to liquid fuels
journal, April 2014


Formaldehyde incorporation by a new methylotroph (L3).
journal, January 1978


Construction of CoA-dependent 1-butanol synthetic pathway functions under aerobic conditions in Escherichia coli
journal, June 2015


Parallel labeling experiments with [U-13C]glucose validate E. coli metabolic network model for 13C metabolic flux analysis
journal, September 2012


Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate
journal, August 2015

  • Leßmeier, Lennart; Pfeifenschneider, Johannes; Carnicer, Marc
  • Applied Microbiology and Biotechnology, Vol. 99, Issue 23
  • DOI: 10.1007/s00253-015-6906-5

Quantifying Biomass Composition by Gas Chromatography/Mass Spectrometry
journal, September 2014

  • Long, Christopher P.; Antoniewicz, Maciek R.
  • Analytical Chemistry, Vol. 86, Issue 19
  • DOI: 10.1021/ac502734e

13C metabolic flux analysis of microbial and mammalian systems is enhanced with GC-MS measurements of glycogen and RNA labeling
journal, November 2016


Characterization of physiological responses to 22 gene knockouts in Escherichia coli central carbon metabolism
journal, September 2016


A modified pathway for the production of acetone in Escherichia coli
journal, January 2013


Measuring the Composition and Stable-Isotope Labeling of Algal Biomass Carbohydrates via Gas Chromatography/Mass Spectrometry
journal, April 2016


Metabolic engineering ofClostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon
journal, November 1993

  • Mermelstein, Lee D.; Papoutsakis, Eleftherios T.; Petersen, Daniel J.
  • Biotechnology and Bioengineering, Vol. 42, Issue 9
  • DOI: 10.1002/bit.260420906

Engineering Escherichia coli for methanol conversion
journal, March 2015


Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria
journal, May 2004


Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress
journal, February 2009


Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol
journal, May 2017

  • Rohlhill, Julia; Sandoval, Nicholas R.; Papoutsakis, Eleftherios T.
  • ACS Synthetic Biology, Vol. 6, Issue 8, p. 1584-1595
  • DOI: 10.1021/acssynbio.7b00114

Automated design of synthetic ribosome binding sites to control protein expression
journal, October 2009

  • Salis, Howard M.; Mirsky, Ethan A.; Voigt, Christopher A.
  • Nature Biotechnology, Vol. 27, Issue 10, p. 946-950
  • DOI: 10.1038/nbt.1568

A new alcohol dehydrogenase, reactive towards methanol, from Bacillus stearothermophilus
journal, June 1988

  • Sheehan, M. C.; Bailey, C. J.; Dowds, B. C. A.
  • Biochemical Journal, Vol. 252, Issue 3
  • DOI: 10.1042/bj2520661

Investigating the effects of perturbations to pgi and eno gene expression on central carbon metabolism in Escherichia coli using 13 C metabolic flux analysis
journal, January 2012

  • Usui, Yuki; Hirasawa, Takashi; Furusawa, Chikara
  • Microbial Cell Factories, Vol. 11, Issue 1
  • DOI: 10.1186/1475-2859-11-87

Overflow Metabolism in Escherichia coli during Steady-State Growth: Transcriptional Regulation and Effect of the Redox Ratio
journal, May 2006


A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris
journal, December 2015


Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli
journal, January 2017


Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization
journal, June 2015

  • Whitaker, William B.; Sandoval, Nicholas R.; Bennett, Robert K.
  • Current Opinion in Biotechnology, Vol. 33
  • DOI: 10.1016/j.copbio.2015.01.007

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism
journal, January 2015

  • Witthoff, Sabrina; Schmitz, Katja; Niedenführ, Sebastian
  • Applied and Environmental Microbiology, Vol. 81, Issue 6, p. 2215-2225
  • DOI: 10.1128/AEM.03110-14

Metabolic Burden: Cornerstones in Synthetic Biology and Metabolic Engineering Applications
journal, August 2016


Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1
journal, February 2016

  • Wu, Tung-Yun; Chen, Chang-Ting; Liu, Jessica Tse-Jin
  • Applied Microbiology and Biotechnology, Vol. 100, Issue 11
  • DOI: 10.1007/s00253-016-7320-3

ePathBrick: A Synthetic Biology Platform for Engineering Metabolic Pathways in E. coli
journal, May 2012

  • Xu, Peng; Vansiri, Amerin; Bhan, Namita
  • ACS Synthetic Biology, Vol. 1, Issue 7
  • DOI: 10.1021/sb300016b

Quantifying Reductive Carboxylation Flux of Glutamine to Lipid in a Brown Adipocyte Cell Line
journal, March 2008

  • Yoo, Hyuntae; Antoniewicz, Maciek R.; Stephanopoulos, Gregory
  • Journal of Biological Chemistry, Vol. 283, Issue 30
  • DOI: 10.1074/jbc.M706494200

Promoters and transcripts for genes involved in methanol oxidation in Methylobacterium extorquens AM1
journal, April 2003


Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR
journal, January 2011


Works referencing / citing this record:

Growth of E. coli on formate and methanol via the reductive glycine pathway
journal, February 2020

  • Kim, Seohyoung; Lindner, Steffen N.; Aslan, Selçuk
  • Nature Chemical Biology, Vol. 16, Issue 5
  • DOI: 10.1038/s41589-020-0473-5

Biological conversion of methane to chemicals and fuels: technical challenges and issues
journal, February 2018

  • Hwang, In Yeub; Nguyen, Anh Duc; Nguyen, Thu Thi
  • Applied Microbiology and Biotechnology, Vol. 102, Issue 7
  • DOI: 10.1007/s00253-018-8842-7

Methanol-essential growth of Escherichia coli
journal, April 2018


Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli
journal, January 2019


C1 Compound Biosensors: Design, Functional Study, and Applications
journal, May 2019

  • Lee, Jin-Young; Sung, Bong Hyun; Oh, So-Hyung
  • International Journal of Molecular Sciences, Vol. 20, Issue 9
  • DOI: 10.3390/ijms20092253

Engineering Artificial Fusion Proteins for Enhanced Methanol Bioconversion
journal, October 2018


Improving formaldehyde consumption drives methanol assimilation in engineered E. coli
journal, June 2018


Charting the Metabolic Landscape of the Facultative Methylotroph Bacillus methanolicus
journal, October 2020


Methanol-essential growth of Escherichia coli
text, January 2018


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