Search Menu
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information
Search Scholarly Publications

Title: Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol

Abstract

In this paper, we report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. Finally, in the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.

Authors:
 [1];  [1];  [2]
+ Show Author Affiliations
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Technical Univ. of Denmark, Horsholm (Denmark)
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); Defense Advanced Research Projects Agency (DARPA) (United States)
OSTI Identifier:
1393096
Grant/Contract Number:  
AC02-05CH11231; HR001134783
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 5; Journal Issue: 12; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; acetone carboxylase; biodegradation; bioengineering; carbon catabolism pathway; sarin; synthetic biology

Citation Formats

Brown, Margaret E., Mukhopadhyay, Aindrila, and Keasling, Jay D. Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol. United States: N. p., 2016. Web. doi:10.1021/acssynbio.6b00115.
Copy to clipboard
Brown, Margaret E., Mukhopadhyay, Aindrila, & Keasling, Jay D. Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol. United States. https://doi.org/10.1021/acssynbio.6b00115
Copy to clipboard
Brown, Margaret E., Mukhopadhyay, Aindrila, and Keasling, Jay D. Tue . "Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol". United States. https://doi.org/10.1021/acssynbio.6b00115. https://www.osti.gov/servlets/purl/1393096.
Copy to clipboard
@article{osti_1393096,
title = {Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol},
author = {Brown, Margaret E. and Mukhopadhyay, Aindrila and Keasling, Jay D.},
abstractNote = {In this paper, we report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. Finally, in the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.},
doi = {10.1021/acssynbio.6b00115},
journal = {ACS Synthetic Biology},
number = 12,
volume = 5,
place = {United States},
year = {Tue Jul 12 00:00:00 EDT 2016},
month = {Tue Jul 12 00:00:00 EDT 2016}
}
Copy to clipboard
Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1021/acssynbio.6b00115
Copyright Statement
Other availability
Search WorldCat Search WorldCat to find libraries that may hold this journal
Citation Metrics:
Cited by: 4 works
Citation information provided by
Web of Science
Save / Share:
Export Metadata
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.

Works referenced in this record:

Production of the antimalarial drug precursor artemisinic acid in engineered yeast
journal, April 2006

  • Ro, Dae-Kyun; Paradise, Eric M.; Ouellet, Mario
  • Nature, Vol. 440, Issue 7086, p. 940-943
  • DOI: 10.1038/nature04640

Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways
journal, February 2011

  • Bond-Watts, Brooks B.; Bellerose, Robert J.; Chang, Michelle C. Y.
  • Nature Chemical Biology, Vol. 7, Issue 4
  • DOI: 10.1038/nchembio.537

Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels
journal, January 2008

  • Atsumi, Shota; Hanai, Taizo; Liao, James C.
  • Nature, Vol. 451, Issue 7174, p. 86-89
  • DOI: 10.1038/nature06450

Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts
journal, January 1998

  • Kealey, J. T.; Liu, L.; Santi, D. V.
  • Proceedings of the National Academy of Sciences, Vol. 95, Issue 2
  • DOI: 10.1073/pnas.95.2.505

Metabolic load and heterologous gene expression
journal, January 1995

Stereoselective Detoxification of Chiral Sarin and Soman Analogues by Phosphotriesterase
journal, August 2001

The environmental fate of organophosphorus nerve agents: A review
journal, February 1995

  • Kingery, Andrew F.; Allen, Herbert E.
  • Toxicological & Environmental Chemistry, Vol. 47, Issue 3-4
  • DOI: 10.1080/02772249509358137

The Structure and Function of a Novel Glycerophosphodiesterase from Enterobacter aerogenes
journal, April 2007

  • Jackson, Colin J.; Carr, Paul D.; Liu, Jian-Wei
  • Journal of Molecular Biology, Vol. 367, Issue 4
  • DOI: 10.1016/j.jmb.2007.01.032

Gene Cloning, Purification, and Characterization of a Phosphodiesterase from Delftia acidovorans
journal, January 2003

Phosphotriesterase variants with high methylphosphonatase activity and strong negative trade-off against phosphotriesters
journal, October 2010

  • Briseño-Roa, Luis; Timperley, Christopher M.; Griffiths, Andrew D.
  • Protein Engineering, Design and Selection, Vol. 24, Issue 1-2
  • DOI: 10.1093/protein/gzq076

Enzymes for the Homeland Defense: Optimizing Phosphotriesterase for the Hydrolysis of Organophosphate Nerve Agents
journal, July 2012

  • Tsai, Ping-Chuan; Fox, Nicholas; Bigley, Andrew N.
  • Biochemistry, Vol. 51, Issue 32
  • DOI: 10.1021/bi300811t

Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway
journal, June 2011

  • Jochimsen, Bjarne; Lolle, Signe; McSorley, Fern R.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 28
  • DOI: 10.1073/pnas.1104922108

Mutational analysis of an Escherichia coli fourteen-gene operon for phosphonate degradation, using TnphoA' elements.
journal, June 1993

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244
journal, July 1983

Design of a secondary alcohol degradation pathway from Pseudomonas fluorescens DSM 50106 in an engineered Escherichia coli
journal, March 2007

  • Kirschner, Anett; Altenbuchner, Josef; Bornscheuer, Uwe T.
  • Applied Microbiology and Biotechnology, Vol. 75, Issue 5
  • DOI: 10.1007/s00253-007-0902-3

PropaneMonooxygenase and NAD + -Dependent Secondary AlcoholDehydrogenase in Propane Metabolism by Gordonia sp.StrainTY-5
journal, December 2003

Novel Acetone Metabolism in a Propane-Utilizing Bacterium, Gordonia sp. Strain TY-5
journal, October 2006

  • Kotani, T.; Yurimoto, H.; Kato, N.
  • Journal of Bacteriology, Vol. 189, Issue 3
  • DOI: 10.1128/JB.01054-06

Purification and characterization of acetone carboxylase from Xanthobacter strain Py2
journal, August 1997

  • Sluis, M. K.; Ensign, S. A.
  • Proceedings of the National Academy of Sciences, Vol. 94, Issue 16
  • DOI: 10.1073/pnas.94.16.8456

Biochemical, Molecular, and Genetic Analyses of the Acetone Carboxylases from Xanthobacter autotrophicus Strain Py2 and Rhodobacter capsulatus Strain B10
journal, June 2002

Bacterial Acetone Carboxylase Is a Manganese-dependent Metalloenzyme
journal, August 2004

  • Boyd, Jeffrey M.; Ellsworth, Heather; Ensign, Scott A.
  • Journal of Biological Chemistry, Vol. 279, Issue 45
  • DOI: 10.1074/jbc.M407177200

The Mechanism of Biotin-Dependent Enzymes
journal, June 1989

BglBrick vectors and datasheets: A synthetic biology platform for gene expression
journal, January 2011

  • Lee, Taek; Krupa, Rachel A.; Zhang, Fuzhong
  • Journal of Biological Engineering, Vol. 5, Issue 1
  • DOI: 10.1186/1754-1611-5-12

Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli
journal, June 2009

  • Bennett, Bryson D.; Kimball, Elizabeth H.; Gao, Melissa
  • Nature Chemical Biology, Vol. 5, Issue 8
  • DOI: 10.1038/nchembio.186

Thiamine Pyrophosphate Stimulates Acetone Activation by Desulfococcus biacutus As Monitored by a Fluorogenic ATP Analogue
journal, May 2014

  • Gutiérrez Acosta, Olga B.; Hardt, Norman; Hacker, Stephan M.
  • ACS Chemical Biology, Vol. 9, Issue 6
  • DOI: 10.1021/cb500152y

Carbonylation as a Key Reaction in Anaerobic Acetone Activation by Desulfococcus biacutus
journal, August 2013

  • Gutiérrez Acosta, Olga B.; Hardt, Norman; Schink, Bernhard
  • Applied and Environmental Microbiology, Vol. 79, Issue 20
  • DOI: 10.1128/AEM.02116-13

Acetone utilization by sulfate-reducing bacteria: draft genome sequence of Desulfococcus biacutus and a proteomic survey of acetone-inducible proteins
journal, July 2014

  • Gutiérrez Acosta, Olga B.; Schleheck, David; Schink, Bernhard
  • BMC Genomics, Vol. 15, Issue 1
  • DOI: 10.1186/1471-2164-15-584

Efficient synthesis of functional isoprenoids from acetoacetate through metabolic pathway-engineered Escherichia coli
journal, January 2009

  • Harada, Hisashi; Yu, Fengnian; Okamoto, Sho
  • Applied Microbiology and Biotechnology, Vol. 81, Issue 5
  • DOI: 10.1007/s00253-008-1724-7

Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm
journal, November 1999

  • Bessette, P. H.; Aslund, F.; Beckwith, J.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 24
  • DOI: 10.1073/pnas.96.24.13703

Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins fromE. coli: To fold or to refold
journal, June 1997

  • Thomas, Jeffrey G.; Ayling, Amanda; Baneyx, François
  • Applied Biochemistry and Biotechnology, Vol. 66, Issue 3
  • DOI: 10.1007/BF02785589

Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli
journal, September 2010

  • Sudhamsu, Jawahar; Kabir, Mariam; Airola, Michael V.
  • Protein Expression and Purification, Vol. 73, Issue 1
  • DOI: 10.1016/j.pep.2010.03.010

Overproduction of theBradyrhizobium japonicum c-Type Cytochrome Subunits of thecbb3Oxidase inEscherichia coli
journal, October 1998

  • Arslan, Engin; Schulz, Henk; Zufferey, Rachel
  • Biochemical and Biophysical Research Communications, Vol. 251, Issue 3
  • DOI: 10.1006/bbrc.1998.9549

Causes and Effects of N-Terminal Codon Bias in Bacterial Genes
journal, September 2013

Codon influence on protein expression in E. coli correlates with mRNA levels
journal, January 2016

  • Boël, Grégory; Letso, Reka; Neely, Helen
  • Nature, Vol. 529, Issue 7586
  • DOI: 10.1038/nature16509

The anti-Shine–Dalgarno sequence drives translational pausing and codon choice in bacteria
journal, March 2012

  • Li, Gene-Wei; Oh, Eugene; Weissman, Jonathan S.
  • Nature, Vol. 484, Issue 7395
  • DOI: 10.1038/nature10965

Avoidance of Truncated Proteins from Unintended Ribosome Binding Sites within Heterologous Protein Coding Sequences
journal, May 2014

  • Whitaker, Weston R.; Lee, Hanson; Arkin, Adam P.
  • ACS Synthetic Biology, Vol. 4, Issue 3
  • DOI: 10.1021/sb500003x

A genome‐scale metabolic reconstruction for Escherichia coli K‐12 MG1655 that accounts for 1260 ORFs and thermodynamic information
journal, January 2007

  • Feist, Adam M.; Henry, Christopher S.; Reed, Jennifer L.
  • Molecular Systems Biology, Vol. 3, Issue 1
  • DOI: 10.1038/msb4100155

Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools
journal, June 2012

  • Ham, T. S.; Dmytriv, Z.; Plahar, H.
  • Nucleic Acids Research, Vol. 40, Issue 18
  • DOI: 10.1093/nar/gks531

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Rapid and Reliable DNA Assembly via Ligase Cycling Reaction
journal, September 2013

  • Kok, Stefan de; Stanton, Leslie H.; Slaby, Todd
  • ACS Synthetic Biology, Vol. 3, Issue 2
  • DOI: 10.1021/sb4001992
    Sort by:
    [ × clear filter / sort ]

    Similar Records in DOE PAGES and OSTI.GOV collections: