Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Metabolic repair through emergence of new pathways in Escherichia coli


Escherichia coli can derive all essential metabolites and cofactors through a highly evolved metabolic system. Damage of pathways may affect cell growth and physiology, but the strategies by which damaged metabolic pathways can be circumvented remain intriguing. Here, we use a ΔpanD (encoding for aspartate 1-decarboxylase) strain of E. coli that is unable to produce the β-alanine required for CoA biosynthesis to demonstrate that metabolic systems can overcome pathway damage by extensively rerouting metabolic pathways and modifying existing enzymes for unnatural functions. Using directed cell evolution, rewiring and repurposing of uracil metabolism allowed formation of an alternative β-alanine biosynthetic pathway. After this pathway was deleted, a second was evolved that used a gain-of-function mutation on ornithine decarboxylase (SpeC) to alter reaction and substrate specificity toward an oxidative decarboxylation–deamination reaction. After deletion of both pathways, yet another independent pathway emerged using polyamine biosynthesis, demonstrating the vast capacity of metabolic repair.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: β-alanine biosynthesis through uracil degradation.
Fig. 2: Characterization of mutations that contribute to the degradation of uracil into β-alanine.
Fig. 3: Emergence of β-alanine synthesis pathway using evolved ornithine decarboxylase.
Fig. 4: β-alanine pathway using SpeC and pathway reconstruction.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. All genomic sequences are available at NCBI under BioProject ID PRJNA485586.


  1. 1.

    Patrick, W. M., Quandt, E. M., Swartzlander, D. B. & Matsumura, I. Multicopy suppression underpins metabolic evolvability. Mol. Biol. Evol. 24, 2716–2722 (2006).

    Article  Google Scholar 

  2. 2.

    Notebaart, R. A., Kintses, B., Feist, A. M. & Papp, B. Underground metabolism: network-level perspective and biotechnological potential. Curr. Opin. Biotechnol. 49, 108–114 (2018).

    CAS  Article  Google Scholar 

  3. 3.

    McLoughlin, S. Y. & Copley, S. D. A compromise required by gene sharing enables survival: implications for evolution of new enzyme activities. Proc. Natl Acad. Sci. USA 105, 13497–13502 (2008).

    CAS  Article  Google Scholar 

  4. 4.

    Blank, D., Wolf, L., Ackermann, M. & Silander, O. K. The predictability of molecular evolution during functional innovation. Proc. Natl Acad. Sci. USA 111, 3044–3049 (2014).

    CAS  Article  Google Scholar 

  5. 5.

    Reed, J. L., Vo, T. D., Schilling, C. H. & Palsson, B. O. An expanded genome-scale model of Escherichia coli K-12 (i JR904 GSM/GPR). genome Biol. 4, R54 (2003).

    Article  Google Scholar 

  6. 6.

    Guzmán, G. I. et al. Model-driven discovery of underground metabolic functions in Escherichia coli. Proc. Natl Acad. Sci. USA 112, 929–934 (2015).

    Article  Google Scholar 

  7. 7.

    Khersonsky, O. & Tawfik, D. S. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu. Rev. Biochem. 79, 471–505 (2010).

    CAS  Article  Google Scholar 

  8. 8.

    Kim, J., Kershner, J. P., Novikov, Y., Shoemaker, R. K. & Copley, S. D. Three serendipitous pathways in E. coli can bypass a block in pyridoxal-5′-phosphate synthesis. Mol. Syst. Biol. 6, 436 (2010).

    CAS  Article  Google Scholar 

  9. 9.

    Webb, M. E., Smith, A. G. & Abell, C. Biosynthesis of pantothenate. Nat. Prod. Rep. 21, 695–721 (2004).

    CAS  Article  Google Scholar 

  10. 10.

    Boldyrev, A. A. Carnosine: new concept for the function of an old molecule. Biochemistry (Mosc). 77, 313–326 (2012).

    CAS  Article  Google Scholar 

  11. 11.

    Joanne, W. M. & Brown, G. M. Purification and properties of l-aspartate-α-decarboxylase, an enzyme that catalyzes the formation of β-alanine in Escherichia coli. J. Biol. Chem. 254, 8074–8082 (1979).

    Google Scholar 

  12. 12.

    Nozaki, S., Webb, M. E. & Niki, H. An activator for pyruvoyl-dependent ʟ-aspartate α-decarboxylase is conserved in a small group of the γ-proteobacteria including Escherichia coli. MicrobiologyOpen 1, 298–310 (2012).

    CAS  Article  Google Scholar 

  13. 13.

    Rathinasabapathi, B. Propionate, a source of β-alanine, is an inhibitor of β-alanine methylation in Limonium latifolium, Plumbaginaceae. J. Plant Physiol. 159, 671–674 (2002).

    CAS  Article  Google Scholar 

  14. 14.

    Fritzson, P. The catabolism of C14-labeled uracil, dihydrouracil, and β-ureidopropionic acid in rat liver slices. J. Biol. Chem. 226, 223–228 (1957).

    CAS  PubMed  Google Scholar 

  15. 15.

    White, W. H., Gunyuzlu, P. L. & Toyn, J. H. Saccharomyces cerevisiae is capable of de novo pantothenic acid biosynthesis involving a novel pathway of beta-alanine production from spermine. J. Biol. Chem. 276, 10794–10800 (2001).

    CAS  Article  Google Scholar 

  16. 16.

    Maruyama, M., Horiuchi, T., Maki, H. & Sekiguchi, M. A dominant (mut D5) and a recessive (dnaQ49) mutator of Escherichia coli. J. Mol. Biol. 167, 757–771 (1983).

    CAS  Article  Google Scholar 

  17. 17.

    Kim, K. S. et al. The Rut pathway for pyrimidine degradation: novel chemistry and toxicity problems. J. Bacteriol. 192, 4089–4102 (2010).

    CAS  Article  Google Scholar 

  18. 18.

    Borodina, I. et al. Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine. Metab. Eng. 27, 57–64 (2015).

    CAS  Article  Google Scholar 

  19. 19.

    Andersen, P. S., Smith, J. M. & Mygind, B. Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12. Eur. J. Biochem. 204, 51–56 (1992).

    CAS  Article  Google Scholar 

  20. 20.

    Bertoldi, M., Carbone, V. & Borri Voltattorni, C. Ornithine and glutamate decarboxylases catalyse an oxidative deamination of their α-methyl substrates. Biochem. J. 342, 509–512 (1999).

    CAS  Article  Google Scholar 

  21. 21.

    Kaminaga, Y. et al. Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation. J. Biol. Chem. 281, 23357–23366 (2006).

    CAS  Article  Google Scholar 

  22. 22.

    Incharoensakdi, A. et al. Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli. Structural and functional properties of wild-type, mutants and E. coli enzymes. Eur. J. Biochem. 267, 7015–7023 (2000).

    CAS  Article  Google Scholar 

  23. 23.

    Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  Google Scholar 

  24. 24.

    Espah Borujeni, A., Channarasappa, A. S. & Salis, H. M. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res. 42, 2646–2659 (2014).

    CAS  Article  Google Scholar 

  25. 25.

    Salis, H. M., Mirsky, E. A. & Voigt, C. A. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946–950 (2009).

    CAS  Article  Google Scholar 

  26. 26.

    Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    CAS  Article  Google Scholar 

  27. 27.

    Degnen, G. E. & Cox, E. C. Conditional mutator gene in Escherichia coli: isolation, mapping, and effector studies. J. Bacteriol. 117, 477–487 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Livingston, D. Deoxyribonucleaic acid polymerase III of Escherichia coli. J. Biol. Chem. 250, 489–497 (1975).

    Google Scholar 

  29. 29.

    Jensen, K. F. & Mygind, B. Different oligomeric states are involved in the allosteric behavior of uracil phosphoribosyltransferase from Escherichia coli. Eur. J. Biochem. 240, 637–645 (1996).

    CAS  Article  Google Scholar 

  30. 30.

    Wernick, D. G., Pontrelli, S. P., Pollock, A. W. & Liao, J. C. Sustainable biorefining in wastewater by engineered extreme alkaliphile Bacillus marmarensis. Sci. Rep. 6, 20224 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Canellakis, E. S., Paterakis, A. A., Huang, S. C., Panagiotidis, C. A. & Kyriakidis, D. A. Identification, cloning, and nucleotide sequencing of the ornithine decarboxylase antizyme gene of Escherichia coli. Proc. Natl Acad. Sci. USA 90, 7129–7133 (1993).

    CAS  Article  Google Scholar 

  32. 32.

    Choi, K. Y., Wernick, D. G., Tat, C. A. & Liao, J. C. Consolidated conversion of protein waste into biofuels and ammonia using Bacillus subtilis. Metab. Eng. 23, 53–61 (2014).

    CAS  Article  Google Scholar 

  33. 33.

    Applebaum, D. M., Dunlap, J. C. & Morris, D. R. Comparison of the biosynthetic and biodegradative ornithine decarboxylases of Escherichia coli. Biochemistry 16, 1580–1584 (1977).

    CAS  Article  Google Scholar 

  34. 34.

    Smart, K. F., Aggio, R. B. M., Van Houtte, J. R. & Villas-Bôas, S. G. Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography-mass spectrometry. Nat. Protoc. 5, 1709–1729 (2010).

    CAS  Article  Google Scholar 

  35. 35.

    Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  Google Scholar 

  36. 36.

    McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

    CAS  Article  Google Scholar 

  37. 37.

    Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strainw1118; iso-2; iso-3. Fly (Austin) 6, 80–92 (2012).

    CAS  Article  Google Scholar 

  38. 38.

    Untergasser, A. et al. Primer3--new capabilities and interfaces. Nucleic Acids Res. 40, e115 (2012).

    CAS  Article  Google Scholar 

  39. 39.

    Liu, M. et al. Global transcriptional programs reveal a carbon source foraging strategy by Escherichia coli. J. Biol. Chem. 280, 15921–15927 (2005).

    CAS  Article  Google Scholar 

Download references


This research was supported in part by a grant from National Science Foundation (MCB-1139318) for JP-US “Metabolomics for Low Carbon Society” (received by J.C.L.), and Japan Science and Technology’s Strategic International Collaborative Research Program (received by E.F). S.F.-G. acknowledges support from a QCB Collaboratory Postdoctoral Fellowship, and the QCB Collaboratory community directed by M. Pellegrini.

Author information




J.C.L. and S.P. conceived the idea of this project and wrote the manuscript. S.P. designed all experiments and performed evolution, RT-qPCR, enzyme assays, gene deletions, genome sequencing library generation, mass spec verification and measured growth phenotypes. R.C.B.F. performed evolution, enzyme assays, point mutation reversions, and growth curve analysis. S.T.T., W.A.L., S.P.P. and E.F. performed metabolomic analysis and data analysis. M.C. performed evolution. S.F.-G. and M.P. performed genomic sequencing and analysis.

Corresponding author

Correspondence to James C. Liao.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–9, Supplementary Tables 1–10

Reporting Summary

Supplementary Dataset 1

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pontrelli, S., Fricke, R.C.B., Teoh, S.T. et al. Metabolic repair through emergence of new pathways in Escherichia coli. Nat Chem Biol 14, 1005–1009 (2018).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing