Discovery of enzymes for toluene synthesis from anoxic microbial communities
Abstract
Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.
- Authors:
-
[1];
[3];
[5];
[2];
[6]
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
- Taipei Medical Univ. (Taiwan)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
- USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Technical Univ. of Denmark, Lyngby (Denmark)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1530356
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature Chemical Biology
- Additional Journal Information:
- Journal Volume: 14; Journal Issue: 5; Journal ID: ISSN 1552-4450
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Beller, Harry R., Rodrigues, Andria V., Zargar, Kamrun, Wu, Yu-Wei, Saini, Avneesh K., Saville, Renee M., Pereira, Jose H., Adams, Paul D., Tringe, Susannah G., Petzold, Christopher J., and Keasling, Jay D. Discovery of enzymes for toluene synthesis from anoxic microbial communities. United States: N. p., 2018.
Web. doi:10.1038/s41589-018-0017-4.
Beller, Harry R., Rodrigues, Andria V., Zargar, Kamrun, Wu, Yu-Wei, Saini, Avneesh K., Saville, Renee M., Pereira, Jose H., Adams, Paul D., Tringe, Susannah G., Petzold, Christopher J., & Keasling, Jay D. Discovery of enzymes for toluene synthesis from anoxic microbial communities. United States. https://doi.org/10.1038/s41589-018-0017-4
Beller, Harry R., Rodrigues, Andria V., Zargar, Kamrun, Wu, Yu-Wei, Saini, Avneesh K., Saville, Renee M., Pereira, Jose H., Adams, Paul D., Tringe, Susannah G., Petzold, Christopher J., and Keasling, Jay D. Mon .
"Discovery of enzymes for toluene synthesis from anoxic microbial communities". United States. https://doi.org/10.1038/s41589-018-0017-4. https://www.osti.gov/servlets/purl/1530356.
@article{osti_1530356,
title = {Discovery of enzymes for toluene synthesis from anoxic microbial communities},
author = {Beller, Harry R. and Rodrigues, Andria V. and Zargar, Kamrun and Wu, Yu-Wei and Saini, Avneesh K. and Saville, Renee M. and Pereira, Jose H. and Adams, Paul D. and Tringe, Susannah G. and Petzold, Christopher J. and Keasling, Jay D.},
abstractNote = {Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.},
doi = {10.1038/s41589-018-0017-4},
journal = {Nature Chemical Biology},
number = 5,
volume = 14,
place = {United States},
year = {Mon Mar 19 00:00:00 EDT 2018},
month = {Mon Mar 19 00:00:00 EDT 2018}
}
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Works referenced in this record:
A prominent glycyl radical enzyme in human gut microbiomes metabolizes trans -4-hydroxy-l-proline
journal, February 2017
- Levin, B. J.; Huang, Y. Y.; Peck, S. C.
- Science, Vol. 355, Issue 6325
FIMO: scanning for occurrences of a given motif
journal, February 2011
- Grant, Charles E.; Bailey, Timothy L.; Noble, William Stafford
- Bioinformatics, Vol. 27, Issue 7
The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4)
journal, October 2015
- Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos
- Standards in Genomic Sciences, Vol. 10, Issue 1
p -Hydroxyphenylacetate decarboxylase from Clostridium difficile : A novel glycyl radical enzyme catalysing the formation of
journal, March 2001
- Selmer, Thorsten; Andrei, Paula I.
- European Journal of Biochemistry, Vol. 268, Issue 5
In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community
journal, August 2016
- Zargar, K.; Saville, R.; Phelan, R. M.
- Scientific Reports, Vol. 6, Issue 1
Circos: An information aesthetic for comparative genomics
journal, June 2009
- Krzywinski, M.; Schein, J.; Birol, I.
- Genome Research, Vol. 19, Issue 9
REFMAC 5 dictionary: organization of prior chemical knowledge and guidelines for its use
journal, November 2004
- Vagin, Alexei A.; Steiner, Roberto A.; Lebedev, Andrey A.
- Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12
Substrate-bound Structures of Benzylsuccinate Synthase Reveal How Toluene Is Activated in Anaerobic Hydrocarbon Degradation
journal, July 2015
- Funk, Michael A.; Marsh, E. Neil G.; Drennan, Catherine L.
- Journal of Biological Chemistry, Vol. 290, Issue 37
From complete genome sequence to ‘complete’ understanding?
journal, August 2010
- Galperin, Michael Y.; Koonin, Eugene V.
- Trends in Biotechnology, Vol. 28, Issue 8
Dual Role for the Tyrosine Decarboxylation Pathway in Enterococcus faecium E17: Response to an Acid Challenge and Generation of a Proton Motive Force
journal, November 2008
- Pereira, C. I.; Matos, D.; San Romao, M. V.
- Applied and Environmental Microbiology, Vol. 75, Issue 2
New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria
journal, January 2005
- Selmer, Thorsten; Pierik, Antonio J.; Heider, Johann
- Biological Chemistry, Vol. 386, Issue 10
Glycyl radical activating enzymes: Structure, mechanism, and substrate interactions
journal, March 2014
- Shisler, Krista A.; Broderick, Joan B.
- Archives of Biochemistry and Biophysics, Vol. 546
Arabidopsis thaliana Nfu2 Accommodates [2Fe-2S] or [4Fe-4S] Clusters and Is Competent for in Vitro Maturation of Chloroplast [2Fe-2S] and [4Fe-4S] Cluster-Containing Proteins
journal, September 2013
- Gao, Huanyao; Subramanian, Sowmya; Couturier, Jérémy
- Biochemistry, Vol. 52, Issue 38
MolProbity: all-atom contacts and structure validation for proteins and nucleic acids
journal, May 2007
- Davis, I. W.; Leaver-Fay, A.; Chen, V. B.
- Nucleic Acids Research, Vol. 35, Issue Web Server
Structural Basis for Allosteric Substrate Specificity Regulation in Anaerobic Ribonucleotide Reductases
journal, August 2001
- Larsson, Karl-Magnus; Andersson, Jessica; Sjöberg, Britt-Marie
- Structure, Vol. 9, Issue 8
Prodigal: prokaryotic gene recognition and translation initiation site identification
journal, March 2010
- Hyatt, Doug; Chen, Gwo-Liang; LoCascio, Philip F.
- BMC Bioinformatics, Vol. 11, Issue 1
Microbial Biosynthesis of Alkanes
journal, July 2010
- Schirmer, A.; Rude, M. A.; Li, X.
- Science, Vol. 329, Issue 5991, p. 559-562
MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets
journal, October 2015
- Wu, Yu-Wei; Simmons, Blake A.; Singer, Steven W.
- Bioinformatics, Vol. 32, Issue 4
CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes
journal, May 2015
- Parks, Donovan H.; Imelfort, Michael; Skennerton, Connor T.
- Genome Research, Vol. 25, Issue 7
Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri.
journal, May 1993
- Molenaar, D.; Bosscher, J. S.; ten Brink, B.
- Journal of Bacteriology, Vol. 175, Issue 10
Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme
journal, November 2012
- Craciun, S.; Balskus, E. P.
- Proceedings of the National Academy of Sciences, Vol. 109, Issue 52
Substrate range of benzylsuccinate synthase from Azoarcus sp. strain T
journal, September 1999
- Beller, Harry R.; Spormann, Alfred M.
- FEMS Microbiology Letters, Vol. 178, Issue 1
UPARSE: highly accurate OTU sequences from microbial amplicon reads
journal, August 2013
- Edgar, Robert C.
- Nature Methods, Vol. 10, Issue 10
Tolumonas auensis gen. nov., sp. nov., a Toluene-Producing Bacterium from Anoxic Sediments of a Freshwater Lake
journal, January 1996
- Fischer-Romero, C.; Tindall, B. J.; Juttner, F.
- International Journal of Systematic Bacteriology, Vol. 46, Issue 1
Three Genomes from the Phylum Acidobacteria Provide Insight into the Lifestyles of These Microorganisms in Soils
journal, February 2009
- Ward, N. L.; Challacombe, J. F.; Janssen, P. H.
- Applied and Environmental Microbiology, Vol. 75, Issue 7
Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees
journal, April 2016
- Letunic, Ivica; Bork, Peer
- Nucleic Acids Research, Vol. 44, Issue W1
RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies
journal, January 2014
- Stamatakis, Alexandros
- Bioinformatics, Vol. 30, Issue 9
The SILVA ribosomal RNA gene database project: improved data processing and web-based tools
journal, November 2012
- Quast, Christian; Pruesse, Elmar; Yilmaz, Pelin
- Nucleic Acids Research, Vol. 41, Issue D1
SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing
journal, May 2012
- Bankevich, Anton; Nurk, Sergey; Antipov, Dmitry
- Journal of Computational Biology, Vol. 19, Issue 5
ezTree: an automated pipeline for identifying phylogenetic marker genes and inferring evolutionary relationships among uncultivated prokaryotic draft genomes
journal, January 2018
- Wu, Yu-Wei
- BMC Genomics, Vol. 19, Issue S1
Styrene biosynthesis from glucose by engineered E. coli
journal, September 2011
- McKenna, Rebekah; Nielsen, David R.
- Metabolic Engineering, Vol. 13, Issue 5
Coot model-building tools for molecular graphics
journal, November 2004
- Emsley, Paul; Cowtan, Kevin
- Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
The COMBREX Project: Design, Methodology, and Initial Results
journal, August 2013
- Anton, Brian P.; Chang, Yi-Chien; Brown, Peter
- PLoS Biology, Vol. 11, Issue 8
Structure and Function of CutC Choline Lyase from Human Microbiota Bacterium Klebsiella pneumoniae
journal, July 2015
- Kalnins, Gints; Kuka, Janis; Grinberga, Solveiga
- Journal of Biological Chemistry, Vol. 290, Issue 35
A critical review of Henry’s law constants for chemicals of environmental interest
journal, October 1981
- Mackay, Donald; Shiu, Wan Ying
- Journal of Physical and Chemical Reference Data, Vol. 10, Issue 4
Anaerobic bacterial metabolism of hydrocarbons
journal, December 1998
- Heider, Johann; Spormann, Alfred M.; Beller, Harry R.
- FEMS Microbiology Reviews, Vol. 22, Issue 5
Protein production by auto-induction in high-density shaking cultures
journal, May 2005
- Studier, F. William
- Protein Expression and Purification, Vol. 41, Issue 1, p. 207-234
Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica : a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism
journal, April 1998
- Leuthner, Birgitta; Leutwein, Christina; Schulz, Henk
- Molecular Microbiology, Vol. 28, Issue 3
Structural Basis for a Kolbe-Type Decarboxylation Catalyzed by a Glycyl Radical Enzyme
journal, September 2011
- Martins, Berta M.; Blaser, Martin; Feliks, Mikolaj
- Journal of the American Chemical Society, Vol. 133, Issue 37
Basic local alignment search tool
journal, October 1990
- Altschul, Stephen F.; Gish, Warren; Miller, Webb
- Journal of Molecular Biology, Vol. 215, Issue 3, p. 403-410
Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities
journal, December 2012
- Akhtar, M. K.; Turner, N. J.; Jones, P. R.
- Proceedings of the National Academy of Sciences, Vol. 110, Issue 1, p. 87-92
Profiling the orphan enzymes
journal, January 2014
- Sorokina, Maria; Stam, Mark; Médigue, Claudine
- Biology Direct, Vol. 9, Issue 1
Catalytic Mechanism of the Glycyl Radical Enzyme 4-Hydroxyphenylacetate Decarboxylase from Continuum Electrostatic and QC/MM Calculations
journal, September 2013
- Feliks, Mikolaj; Martins, Berta M.; Ullmann, G. Matthias
- Journal of the American Chemical Society, Vol. 135, Issue 39
Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega
journal, January 2011
- Sievers, Fabian; Wilm, Andreas; Dineen, David
- Molecular Systems Biology, Vol. 7, Issue 1
The Enzyme Function Initiative
journal, November 2011
- Gerlt, John A.; Allen, Karen N.; Almo, Steven C.
- Biochemistry, Vol. 50, Issue 46
Anoxic hypolimnion is a significant source of biogenic toluene
journal, October 1986
- Jüttner, F.; Henatsch, J. J.
- Nature, Vol. 323, Issue 6091
SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information
journal, April 2014
- Biasini, Marco; Bienert, Stefan; Waterhouse, Andrew
- Nucleic Acids Research, Vol. 42, Issue W1
Assessing the role of p-cresol tolerance in Clostridium difficile
journal, June 2008
- Dawson, L. F.; Stabler, R. A.; Wren, B. W.
- Journal of Medical Microbiology, Vol. 57, Issue 6, p. 745-749
MUSCLE: multiple sequence alignment with high accuracy and high throughput
journal, March 2004
- Edgar, R. C.
- Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797
FastTree: Computing Large Minimum Evolution Trees with Profiles instead of a Distance Matrix
journal, April 2009
- Price, M. N.; Dehal, P. S.; Arkin, A. P.
- Molecular Biology and Evolution, Vol. 26, Issue 7
Anaerobic metabolism of l -phenylalanine via benzoyl-CoA in the denitrifying bacterium Thauera aromatica
journal, September 1997
- Schneider, Sabine; Mohamed, M. E. -S.; Fuchs, G.
- Archives of Microbiology, Vol. 168, Issue 4
Insight into the Mechanism of the B 12 -Independent Glycerol Dehydratase from Clostridium butyricum : Preliminary Biochemical and Structural Characterization ‡
journal, April 2004
- O'Brien, Jessica Rae; Raynaud, Celine; Croux, Christian
- Biochemistry, Vol. 43, Issue 16
4-Hydroxyphenylacetate Decarboxylases: Properties of a Novel Subclass of Glycyl Radical Enzyme Systems †
journal, August 2006
- Yu, Lihua; Blaser, Martin; Andrei, Paula I.
- Biochemistry, Vol. 45, Issue 31
Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View
journal, March 2009
- Carmona, M.; Zamarro, M. T.; Blazquez, B.
- Microbiology and Molecular Biology Reviews, Vol. 73, Issue 1
An optimized growth medium for increased recombinant protein secretion titer via the type III secretion system
journal, February 2021
- Burdette, Lisa Ann; Wong, Han Teng; Tullman-Ercek, Danielle
- Microbial Cell Factories, Vol. 20, Issue 1
Works referencing / citing this record:
Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene‐Producing Glycyl Radical Enzyme
journal, November 2019
- Rodrigues, Andria V.; Tantillo, Dean J.; Mukhopadhyay, Aindrila
- ChemBioChem, Vol. 21, Issue 5
Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals: Emerging tools, enabling technologies, and future opportunities
journal, August 2018
- Machas, Michael; Kurgan, Gavin; Jha, Amit K.
- Journal of Chemical Technology & Biotechnology, Vol. 94, Issue 1