Article

A metabolic pathway for catabolizing levulinic acid in bacteria

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Abstract

Microorganisms can catabolize a wide range of organic compounds and therefore have the potential to perform many industrially relevant bioconversions. One barrier to realizing the potential of biorefining strategies lies in our incomplete knowledge of metabolic pathways, including those that can be used to assimilate naturally abundant or easily generated feedstocks. For instance, levulinic acid (LA) is a carbon source that is readily obtainable as a dehydration product of lignocellulosic biomass and can serve as the sole carbon source for some bacteria. Yet, the genetics and structure of LA catabolism have remained unknown. Here, we report the identification and characterization of a seven-gene operon that enables LA catabolism in Pseudomonas putida KT2440. When the pathway was reconstituted with purified proteins, we observed the formation of four acyl-CoA intermediates, including a unique 4-phosphovaleryl-CoA and the previously observed 3-hydroxyvaleryl-CoA product. Using adaptive evolution, we obtained a mutant of Escherichia coli LS5218 with functional deletions of fadE and atoC that was capable of robust growth on LA when it expressed the five enzymes from the P. putida operon. This discovery will enable more efficient use of biomass hydrolysates and metabolic engineering to develop bioconversions using LA as a feedstock.

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Acknowledgements

Work in the Pfleger laboratory was funded by the National Science Foundation (CBET-114678) and the William F. Vilas Trust. Work in the Deutschbauer and Arkin laboratories was funded by ENIGMA, a Scientific Focus Area Program, supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research and Genomics: GTLFoundational Science through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. Work in the Amador-Noguez laboratory was funded by the HHMI International Student Research Fellowship. R.L.C. was supported by the NIH NHGRI Genomic Sciences Training Program (T32 HG002760). A.L.M. was supported by an NSF SEES fellowship (GEO-1215871). J.M.R. was supported by an NSF Graduate Research Fellowship (DGE-1256259).

The authors thank J. Escalante for providing plasmid pK18mobsacB and J. Altenbuchner for providing strain P. putida KTU and plasmid pJOE6261.2. The authors acknowledge the Mass Spectrometry/Proteomics Facility at the UW–Madison Biotechnology Center for performing the in-gel digest and providing the LC–MS/MS results, and the UW–Madison Biotechnology Center DNA Sequencing Facility for providing genomic sequencing services. The authors also thank G. Gordon for help with the analysis of the E. coli genomic sequencing single nucleotide polymorphisms (SNPs).

Author information

Affiliations

  1. Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, 53706, USA

    • Jacqueline M. Rand
    • , Ryan L. Clark
    • , Joshua M. Thiede
    • , Christopher R. Mehrer
    • , Daniel E. Agnew
    • , Candace E. Campbell
    • , Andrew L. Markley
    •  & Brian F. Pfleger
  2. Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI, 53706, USA

    • Tippapha Pisithkul
    •  & Daniel Amador-Noguez
  3. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA

    • Morgan N. Price
    • , Jayashree Ray
    • , Kelly M. Wetmore
    • , Yumi Suh
    • , Adam P. Arkin
    •  & Adam M. Deutschbauer
  4. Department of Bioengineering, University of California, Berkeley, CA, 94720, USA

    • Adam P. Arkin
  5. Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, 53706, USA

    • Daniel Amador-Noguez

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Contributions

J.M.R., D.E.A. and B.F.P. conceived the study. J.M.R. designed and performed the experiments and analysed the data, with the following exceptions. T.P. and D.A.-N. designed the LC–MS/MS experiments and T.P. performed the LC–MS and LC–MS/MS experiments. D.E.A. and J.M.T. performed the transposon library screen. C.E.C. assisted with the promoter and CoA ligase assay. A.L.M. proposed, and helped design and perform, the pulldown experiment. Y.S. and J.R. prepared the RB-TnSeq mutant library of P. putida KT2440 (Putida_ML5). K.M.W., R.L.C., J.R. and A.M.D. performed the fitness assays with the Putida_ML5 library. M.N.P. performed the data analysis to determine fitness values. R.L.C. prepared the supplementary analysis of the Putida_ML5 fitness experiments. A.M.D. and A.P.A. managed the Bar-Seq experiments. C.R.M helped design and analyse the prevalence of the lva operon in other organisms. J.M.R. and B.F.P. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Brian F. Pfleger.

Electronic supplementary material

  1. Supplementary Information

    Supplementary Notes, Supplementary References, Supplementary Figures 1–6, Supplementary Tables 1–4, Supplementary Tables 7–10.

  2. Life Sciences Reporting Summary

  3. Supplementary Table 5 and 6

    Species with LvaABCD homologues and Species with LvaACD homologues.

  4. Supplementary Data Set 1

    Data code.