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Discovery of enzymes for toluene synthesis from anoxic microbial communities

Nature Chemical Biology volume 14, pages 451–457 (2018)Cite this article

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.

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Fig. 1: Glycyl radical enzymes encoded in the toluene-producing sewage culture metagenome and their association with in vitro phenylacetate decarboxylase activity.
Fig. 2: Homologous phenylacetate decarboxylase gene clusters from sewage and lake sediment cultures.
Fig. 3: Reactions catalyzed by PhdA and PhdB.
Fig. 4: Multiple-sequence alignments comparing PhdB and PhdA with other glycyl radical enzymes and glycyl radical activating enzymes, respectively.
Fig. 5: Characterization of the putatively toluene-producing Acidobacteria strain Tolsyn based on its recovered genome.

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Acknowledgements

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.

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Author notes

  1. These authors contributed equally: Harry R. Beller, Andria V. Rodrigues, Kamrun Zargar.

Authors and Affiliations

  1. Joint BioEnergy Institute (JBEI), Emeryville, CA, USA

    Harry R. Beller, Andria V. Rodrigues, Kamrun Zargar, Avneesh K. Saini, Renee M. Saville, Jose H. Pereira, Paul D. Adams, Christopher J. Petzold & Jay D. Keasling

  2. Earth and Environmental Sciences, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA, USA

    Harry R. Beller

  3. Graduate Institute of Biomedical Informatics, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan

    Yu-Wei Wu

  4. Molecular Biophysics & Integrated Bioimaging, LBNL, Berkeley, CA, USA

    Jose H. Pereira & Paul D. Adams

  5. Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA

    Paul D. Adams & Jay D. Keasling

  6. Joint Genome Institute, Walnut Creek, CA, USA

    Susannah G. Tringe

  7. Biological Systems and Engineering, LBNL, Berkeley, CA, USA

    Christopher J. Petzold & Jay D. Keasling

  8. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA

    Jay D. Keasling

  9. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark

    Jay D. Keasling

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Contributions

H.R.B., A.V.R., K.Z., and R.M.S. conceived of and designed the experiments. A.V.R. (primarily) and A.K.S. conducted recombinant protein studies, K.Z. performed activity-based protein fractionation, R.M.S. cultivated lake sediment cultures, and H.R.B. assisted with all types of experiments. H.R.B., Y.-W.W., and A.V.R. analyzed the data. S.G.T. oversaw the production of metagenomic data, and C.J.P. oversaw the production of metaproteomic data. J.H.P. and P.D.A. performed molecular modeling analyses of PhdB. The manuscript was written by H.R.B. (primarily), and all other authors, including J.D.K., contributed to refinement of the text.

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Correspondence to Harry R. Beller.

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J.D.K. has a financial interest in Amyris, Lygos, Demetrix, and Constructive Biology.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Tables 1 and 2 and Supplementary Note 1

Life Sciences Reporting Summary

Supplementary Dataset 1

Shotgun proteomic data for FPLC fractions

Supplementary Dataset 2

Community composition for lake sediment culture

Supplementary Dataset 3

Community composition for sewage culture

Supplementary Dataset 4

JGI metagenome metadata and methods summary

Supplementary Dataset 5

Newick file for Fig. 1

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Beller, H.R., Rodrigues, A.V., Zargar, K. et al. Discovery of enzymes for toluene synthesis from anoxic microbial communities. Nat Chem Biol 14, 451–457 (2018). https://doi.org/10.1038/s41589-018-0017-4

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