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.
Subscribe to Journal
Get full journal access for 1 year
only $14.08 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.
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.
Patrick, W. M., Quandt, E. M., Swartzlander, D. B. & Matsumura, I. Multicopy suppression underpins metabolic evolvability. Mol. Biol. Evol. 24, 2716–2722 (2006).
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).
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).
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).
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).
Guzmán, G. I. et al. Model-driven discovery of underground metabolic functions in Escherichia coli. Proc. Natl Acad. Sci. USA 112, 929–934 (2015).
Khersonsky, O. & Tawfik, D. S. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu. Rev. Biochem. 79, 471–505 (2010).
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).
Webb, M. E., Smith, A. G. & Abell, C. Biosynthesis of pantothenate. Nat. Prod. Rep. 21, 695–721 (2004).
Boldyrev, A. A. Carnosine: new concept for the function of an old molecule. Biochemistry (Mosc). 77, 313–326 (2012).
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).
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).
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).
Fritzson, P. The catabolism of C14-labeled uracil, dihydrouracil, and β-ureidopropionic acid in rat liver slices. J. Biol. Chem. 226, 223–228 (1957).
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).
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).
Kim, K. S. et al. The Rut pathway for pyrimidine degradation: novel chemistry and toxicity problems. J. Bacteriol. 192, 4089–4102 (2010).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Degnen, G. E. & Cox, E. C. Conditional mutator gene in Escherichia coli: isolation, mapping, and effector studies. J. Bacteriol. 117, 477–487 (1974).
Livingston, D. Deoxyribonucleaic acid polymerase III of Escherichia coli. J. Biol. Chem. 250, 489–497 (1975).
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).
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).
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).
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).
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).
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).
Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).
McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).
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).
Untergasser, A. et al. Primer3--new capabilities and interfaces. Nucleic Acids Res. 40, e115 (2012).
Liu, M. et al. Global transcriptional programs reveal a carbon source foraging strategy by Escherichia coli. J. Biol. Chem. 280, 15921–15927 (2005).
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.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
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). https://doi.org/10.1038/s41589-018-0149-6
Curating a comprehensive set of enzymatic reaction rules for efficient novel biosynthetic pathway design
Metabolic Engineering (2021)
Trends in Biotechnology (2020)
The FEBS Journal (2020)
Annual Review of Microbiology (2020)
Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds
Metabolic Engineering (2020)