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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $15.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Galperin, M. Y. & Koonin, E. V. From complete genome sequence to ‘complete’ understanding? Trends Biotechnol. 28, 398–406 (2010).
Gerlt, J. A. et al. The Enzyme Function Initiative. Biochemistry 50, 9950–9962 (2011).
Anton, B. P. et al. The COMBREX project: design, methodology, and initial results. PLoS Biol. 11, e1001638 (2013).
Lespinet, O. & Labedan, B. Orphan enzymes? Science 307, 42 (2005).
Sorokina, M., Stam, M., Médigue, C., Lespinet, O. & Vallenet, D. Profiling the orphan enzymes. Biol. Direct 9, 10 (2014).
McKenna, R. & Nielsen, D. R. Styrene biosynthesis from glucose by engineered E. coli. Metab. Eng. 13, 544–554 (2011).
Jüttner, F. & Henatsch, J. J. Anoxic hypolimnion is a significant source of biogenic toluene. Nature 323, 797–798 (1986).
Zargar, K. et al. In vitro characterization of phenylacetate decarboxylase, a novel enzyme catalyzing toluene biosynthesis in an anaerobic microbial community. Sci. Rep. 6, 31362 (2016).
Fischer-Romero, C., Tindall, B. J. & Jüttner, F. Tolumonas auensis gen. nov., sp. nov., a toluene-producing bacterium from anoxic sediments of a freshwater lake. Int. J. Syst. Bacteriol. 46, 183–188 (1996).
Pons, J. L., Rimbault, A., Darbord, J. C. & Leluan, G. [Biosynthesis of toluene in Clostridium aerofoetidu m strain WS]. Ann. Microbiol. (Paris) 135B, 219–222 (1984).
Akhtar, M. K., Turner, N. J. & Jones, P. R. Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities. Proc. Natl. Acad. Sci. USA 110, 87–92 (2013).
Schirmer, A., Rude, M. A., Li, X., Popova, E. & del Cardayre, S. B. Microbial biosynthesis of alkanes. Science 329, 559–562 (2010).
Selmer, T. & Andrei, P. I. p-Hydroxyphenylacetate decarboxylase from Clostridium difficile. A novel glycyl radical enzyme catalysing the formation of p-cresol. Eur. J. Biochem. 268, 1363–1372 (2001).
Yu, L., Blaser, M., Andrei, P. I., Pierik, A. J. & Selmer, T. 4-Hydroxyphenylacetate decarboxylases: properties of a novel subclass of glycyl radical enzyme systems. Biochemistry 45, 9584–9592 (2006).
Selmer, T., Pierik, A. J. & Heider, J. New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria. Biol. Chem. 386, 981–988 (2005).
Shisler, K. A. & Broderick, J. B. Glycyl radical activating enzymes: structure, mechanism, and substrate interactions. Arch. Biochem. Biophys. 546, 64–71 (2014).
Leuthner, B. et al. Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism. Mol. Microbiol. 28, 615–628 (1998).
O’Brien, J. R. et al. Insight into the mechanism of the B12-independent glycerol dehydratase from Clostridium butyricum: preliminary biochemical and structural characterization. Biochemistry 43, 4635–4645 (2004).
Beller, H. R. & Spormann, A. M. Substrate range of benzylsuccinate synthase from Azoarcus sp. strain T. FEMS Microbiol. Lett. 178, 147–153 (1999).
Becker, A. et al. Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase. Nat. Struct. Biol. 6, 969–975 (1999).
Larsson, K. M., Andersson, J., Sjöberg, B. M., Nordlund, P. & Logan, D. T. Structural basis for allosteric substrate specificity regulation in anaerobic ribonucleotide reductases. Structure 9, 739–750 (2001).
Heider, J., Spormann, A. M., Beller, H. R. & Widdel, F. Anaerobic bacterial metabolism of hydrocarbons. FEMS Microbiol. Rev. 22, 459–473 (1998).
Feliks, M., Martins, B. M. & Ullmann, G. M. Catalytic mechanism of the glycyl radical enzyme 4-hydroxyphenylacetate decarboxylase from continuum electrostatic and QC/MM calculations. J. Am. Chem. Soc. 135, 14574–14585 (2013).
Kalnins, G. et al. Structure and function of CutC choline lyase from human microbiota bacterium Klebsiella pneumoniae. J. Biol. Chem. 290, 21732–21740 (2015).
Craciun, S. & Balskus, E. P. Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme. Proc. Natl. Acad. Sci. USA 109, 21307–21312 (2012).
Levin, B. J. et al. A prominent glycyl radical enzyme in human gut microbiomes metabolizes trans-4-hydroxy-l-proline. Science 355, eaai8386 (2017).
Funk, M. A., Marsh, E. N. & Drennan, C. L. Substrate-bound structures of benzylsuccinate synthase reveal how toluene is activated in anaerobic hydrocarbon degradation. J. Biol. Chem. 290, 22398–22408 (2015).
Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).
Martins, B. M. et al. Structural basis for a Kolbe-type decarboxylation catalyzed by a glycyl radical enzyme. J. Am. Chem. Soc. 133, 14666–14674 (2011).
Kielak, A. M., Barreto, C. C., Kowalchuk, G. A., van Veen, J. A. & Kuramae, E. E. The ecology of Acidobacteria: moving beyond genes and genomes. Front. Microbiol. 7, 744 (2016).
Ward, N. L. et al. Three genomes from the phylum Acidobacteria provide insight into the lifestyles of these microorganisms in soils. Appl. Environ. Microbiol. 75, 2046–2056 (2009).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Dawson, L. F., Stabler, R. A. & Wren, B. W. Assessing the role of p-cresol tolerance in Clostridium difficile. J. Med. Microbiol. 57, 745–749 (2008).
Schneider, S., Mohamed, M. E. S. & Fuchs, G. Anaerobic metabolism of L-phenylalanine via benzoyl-CoA in the denitrifying bacterium Thauera aromatica. Arch. Microbiol. 168, 310–320 (1997).
Carmona, M. et al. Anaerobic catabolism of aromatic compounds: a genetic and genomic view. Microbiol. Mol. Biol. Rev. 73, 71–133 (2009).
Molenaar, D., Bosscher, J. S., ten Brink, B., Driessen, A. J. & Konings, W. N. Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri. J. Bacteriol. 175, 2864–2870 (1993).
Pereira, C. I., Matos, D., San Romão, M. V. & Crespo, M. T. Dual role for the tyrosine decarboxylation pathway in Enterococcus faecium E17: response to an acid challenge and generation of a proton motive force. Appl. Environ. Microbiol. 75, 345–352 (2009).
Beller, H. R., Legler, T. C. & Kane, S. R. Genetic manipulation of the obligate chemolithoautotrophic bacterium Thiobacillus denitrificans. Methods Mol. Biol. 881, 99–136 (2012).
Huntemann, M. et al. The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4). Stand. Genomic Sci. 10, 86 (2015).
Edgar, R. C. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10, 996–998 (2013).
Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590–D596 (2013).
Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).
Gao, H. et al. 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. Biochemistry 52, 6633–6645 (2013).
Mackay, D. & Shiu, W. Y. A critical review of Henry’s Law constants for chemicals of environmental interest. J. Phys. Chem. Ref. Data 10, 1175–1199 (1981).
Grant, C. E., Bailey, T. L. & Noble, W. S. FIMO: scanning for occurrences of a given motif. Bioinformatics 27, 1017–1018 (2011).
Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).
Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).
Letunic, I. & Bork, P. Interactive tree of life (iTOL)v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 44, W242–W245 (2016).
Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477 (2012).
Wu, Y. W., Simmons, B. A. & Singer, S. W. MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets. Bioinformatics 32, 605–607 (2016).
Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11, 119 (2010).
Parks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P. & Tyson, G. W. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 25, 1043–1055 (2015).
Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).
Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26, 1641–1650 (2009).
Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Vagin, A. A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D Biol. Crystallogr. 60, 2184–2195 (2004).
Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).
Wu, Y.-W. ezTree: an automated pipeline for identifying phylogenetic marker genes and inferring evolutionary relationships among uncultivated prokaryotic draft genomes. BMC Genomics 19, 921 (2018).
We thank the following people from JBEI, LBNL, and JGI for their valuable contributions to this work: U. Karaoz, N. Hillson, A. DeGiovanni, E.-B. Goh, E. Baidoo, X. Wang, S. Wang, P. Sorensen, S. Yilmaz, G. Goyal, J. Heazlewood, T. Glavina del Rio, S. Malfatti, E. Eloe-Fadrosh, A. Rivers, and G. Tomaleri. We also thank M. Salemi (UC Davis Genome Center, Proteomics Core Facility). This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org), supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. Work conducted by the Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231.
Supplementary Figures 1–12, Supplementary Tables 1 and 2 and Supplementary Note 1
Shotgun proteomic data for FPLC fractions
Community composition for lake sediment culture
Community composition for sewage culture
JGI metagenome metadata and methods summary
Newick file for Fig. 1
About this article
Indoleacetate decarboxylase is a glycyl radical enzyme catalysing the formation of malodorant skatole
Nature Communications (2018)