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Title: Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli

Abstract

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. In conclusion, by incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.

Authors:
 [1];  [2];  [1];  [3];  [4];  [4];  [3];  [3];  [3];  [5];  [1]
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  1. Univ. of Delaware, Newark, DE (United States). Dept. of Chemical and Biomolecular Engineering; Univ. of Delaware, Newark, DE (United States). Delaware Biotechnology Inst.
  2. Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies; Hamilton College, Clinton, NY (United States)
  3. Univ. of Delaware, Newark, DE (United States). Dept. of Chemical and Biomolecular Engineering
  4. Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies
  5. Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies; Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies
Publication Date:
Research Org.:
Univ. of Delaware, Newark, DE (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
OSTI Identifier:
1410784
Grant/Contract Number:  
AR0000432
Resource Type:
Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 39; Journal Issue: C; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Methylotrophy; Methanol; E. coli; Pathway engineering for substrate utilization

Citation Formats

Whitaker, W. Brian, Jones, J. Andrew, Bennett, R. Kyle, Gonzalez, Jacqueline E., Vernacchio, Victoria R., Collins, Shannon M., Palmer, Michael A., Schmidt, Samuel, Antoniewicz, Maciek R., Koffas, Mattheos A., and Papoutsakis, Eleftherios T. Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli. United States: N. p., 2016. Web. doi:10.1016/j.ymben.2016.10.015.
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Whitaker, W. Brian, Jones, J. Andrew, Bennett, R. Kyle, Gonzalez, Jacqueline E., Vernacchio, Victoria R., Collins, Shannon M., Palmer, Michael A., Schmidt, Samuel, Antoniewicz, Maciek R., Koffas, Mattheos A., & Papoutsakis, Eleftherios T. Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli. United States. https://doi.org/10.1016/j.ymben.2016.10.015
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Whitaker, W. Brian, Jones, J. Andrew, Bennett, R. Kyle, Gonzalez, Jacqueline E., Vernacchio, Victoria R., Collins, Shannon M., Palmer, Michael A., Schmidt, Samuel, Antoniewicz, Maciek R., Koffas, Mattheos A., and Papoutsakis, Eleftherios T. Tue . "Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli". United States. https://doi.org/10.1016/j.ymben.2016.10.015. https://www.osti.gov/servlets/purl/1410784.
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@article{osti_1410784,
title = {Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli},
author = {Whitaker, W. Brian and Jones, J. Andrew and Bennett, R. Kyle and Gonzalez, Jacqueline E. and Vernacchio, Victoria R. and Collins, Shannon M. and Palmer, Michael A. and Schmidt, Samuel and Antoniewicz, Maciek R. and Koffas, Mattheos A. and Papoutsakis, Eleftherios T.},
abstractNote = {Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. In conclusion, by incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.},
doi = {10.1016/j.ymben.2016.10.015},
journal = {Metabolic Engineering},
number = C,
volume = 39,
place = {United States},
year = {Tue Nov 01 00:00:00 EDT 2016},
month = {Tue Nov 01 00:00:00 EDT 2016}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1016/j.ymben.2016.10.015
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Cited by: 121 works
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Figures / Tables:

Figure 1 Figure 1: Methanol utilization in E. coli. Enzymes required for the assimilation of methanol into central metabolism are shown in red: MDH (methanol dehydrogenase), HPS (3-hexulose-6-phosphate synthase) and PHI (6-phospho-3-hexuloisomerase). Methanol is oxidized to formaldehyde, which is fixed to the pentose phosphate pathway intermediate, ribulose-5-phosphate (Ru5P), yielding hexulose-6-phosphate (H6P). H6Pmore » is then isomerized to fructose-6-phosphate (F6P), which can enter the Embden–Meyerhof–Parnas (EMP) pathway to yield pyruvate and acetyl-CoA for metabolite production or the pentose phosphate pathway to regenerate Ru5P. The native formaldehyde detoxification pathway (formaldehyde to CO2) has been disrupted by knocking out formaldehyde dehydrogenase (frmA).« less
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    Works referencing / citing this record:

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