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Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol


1,4-Butanediol (BDO) is an important commodity chemical used to manufacture over 2.5 million tons annually of valuable polymers, and it is currently produced exclusively through feedstocks derived from oil and natural gas. Herein we report what are to our knowledge the first direct biocatalytic routes to BDO from renewable carbohydrate feedstocks, leading to a strain of Escherichia coli capable of producing 18 g l−1 of this highly reduced, non-natural chemical. A pathway-identification algorithm elucidated multiple pathways for the biosynthesis of BDO from common metabolic intermediates. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic operation of the oxidative tricarboxylic acid cycle, thereby generating reducing power to drive the BDO pathway. The organism produced BDO from glucose, xylose, sucrose and biomass-derived mixed sugar streams. This work demonstrates a systems-based metabolic engineering approach to strain design and development that can enable new bioprocesses for commodity chemicals that are not naturally produced by living cells.

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Figure 1: Overview of the Biopathway Predictor network calculation and analysis procedure.
Figure 2: Production of BDO from glucose in engineered E. coli strains.
Figure 3: Flux distribution of ECKh-422 pZS*13S-sucCD-sucD-4hbd/sucA pZE23-002C-Cat2 determined by 13C metabolic flux analysis.
Figure 4: Production of BDO from various carbohydrate sources.
Figure 5: Production of BDO from glucose in 2-l fed-batch fermentation using the OptKnock strain ECKh-422.


  1. Carole, T.M., Pellegrino, J. & Paster, M.D. Opportunities in the industrial biobased products industry. Appl. Biochem. Biotechnol. 113–116, 871–885 (2004).

    Article  Google Scholar 

  2. Kroschwitz, J.I. & Howe-Grant, M. Encyclopedia of Chemical Technology (John Wiley and Sons, New York, 1993).

  3. Nakamura, C.E. & Whited, G.M. Metabolic engineering for the microbial production of 1,3-propanediol. Curr. Opin. Biotechnol. 14, 454–459 (2003).

    Article  CAS  Google Scholar 

  4. Altaras, N.E. & Cameron, D.C. Metabolic engineering of a 1,2-propanediol pathway in Escherichia coli. Appl. Environ. Microbiol. 65, 1180–1185 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Atsumi, S. et al. Metabolic engineering of Escherichia coli for 1-butanol production. Metab. Eng. 10, 305–311 (2008).

    Article  CAS  Google Scholar 

  6. Lindberg, P., Park, S. & Melis, A. Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism. Metab. Eng. 12, 70–79 (2010).

    Article  CAS  Google Scholar 

  7. Qian, Z.G., Xia, X.X. & Lee, S.Y. Metabolic engineering of Escherichia coli for the production of putrescine: a four carbon diamine. Biotechnol. Bioeng. 104, 651–662 (2009).

    CAS  PubMed  Google Scholar 

  8. Lee, S.J., Song, H. & Lee, S.Y. Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production. Appl. Environ. Microbiol. 72, 1939–1948 (2006).

    Article  CAS  Google Scholar 

  9. Moon, T.S., Yoon, S.H., Lanza, A.M., Roy-Mayhew, J.D. & Prather, K.L. Production of glucaric acid from a synthetic pathway in recombinant Escherichia coli. Appl. Environ. Microbiol. 75, 589–595 (2009).

    Article  CAS  Google Scholar 

  10. Rathnasingh, C., Raj, S.M., Jo, J.E. & Park, S. Development and evaluation of efficient recombinant Escherichia coli strains for the production of 3-hydroxypropionic acid from glycerol. Biotechnol. Bioeng. 104, 729–739 (2009).

    CAS  PubMed  Google Scholar 

  11. Lee, K.H., Park, J.H., Kim, T.Y., Kim, H.U. & Lee, S.Y. Systems metabolic engineering of Escherichia coli for L-threonine production. Mol. Syst. Biol. 3, 149 (2007).

    Article  CAS  Google Scholar 

  12. Park, J.H., Lee, K.H., Kim, T.Y. & Lee, S.Y. Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation. Proc. Natl. Acad. Sci. USA 104, 7797–7802 (2007).

    Article  CAS  Google Scholar 

  13. Hong, S.H. & Lee, S.Y. Metabolic flux analysis for succinic acid production by recombinant Escherichia coli with amplified malic enzyme activity. Biotechnol. Bioeng. 74, 89–95 (2001).

    Article  CAS  Google Scholar 

  14. Reed, J.L., Vo, T.D., Schilling, C.H. & Palsson, B.O. An expanded genome-scale model of Escherichia coli K-12 (iJR904 GSM/GPR). Genome Biol. 4, R54 (2003).

    Article  Google Scholar 

  15. Cho, A., Yun, H., Park, J.H., Lee, S.Y. & Park, S. Prediction of novel synthetic pathways for the production of desired chemicals. BMC Syst. Biol. 4, 35 (2010).

    Article  Google Scholar 

  16. Hatzimanikatis, V. et al. Exploring the diversity of complex metabolic networks. Bioinformatics 21, 1603–1609 (2005).

    Article  CAS  Google Scholar 

  17. Henry, C.S., Broadbelt, L.J. & Hatzimanikatis, V. Discovery and analysis of novel metabolic pathways for the biosynthesis of industrial chemicals: 3-hydroxypropanoate. Biotechnol. Bioeng. 106, 462–473 (2010).

    CAS  PubMed  Google Scholar 

  18. Mavrovouniotis, M.L. Group contributions for estimating standard gibbs energies of formation of biochemical compounds in aqueous solution. Biotechnol. Bioeng. 36, 1070–1082 (1990).

    Article  CAS  Google Scholar 

  19. Burk, M.B., Burgard, A.P., Osterhout, R.E. & Sun, J. Microorganisms for the production of 1,4-butanediol. International Bureau patent WO/2010/030711 (2010).

  20. Saito, N. et al. Metabolite profiling reveals YihU as a novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde metabolism in Escherichia coli. J. Biol. Chem. 284, 16442–16451 (2009).

    Article  CAS  Google Scholar 

  21. Söhling, B. & Gottschalk, G. Purification and characterization of a coenzyme-A-dependent succinate-semialdehyde dehydrogenase from Clostridium kluyveri. Eur. J. Biochem. 212, 121–127 (1993).

    Article  Google Scholar 

  22. Söhling, B. & Gottschalk, G. Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri. J. Bacteriol. 178, 871–880 (1996).

    Article  Google Scholar 

  23. Valentin, H.E., Zwingmann, G., Schonebaum, A. & Steinbuchel, A. Metabolic pathway for biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from 4-hydroxybutyrate by Alcaligenes eutrophus. Eur. J. Biochem. 227, 43–60 (1995).

    Article  CAS  Google Scholar 

  24. Tian, J., Bryk, R., Itoh, M., Suematsu, M. & Nathan, C. Variant tricarboxylic acid cycle in Mycobacterium tuberculosis: identification of alpha-ketoglutarate decarboxylase. Proc. Natl. Acad. Sci. USA 102, 10670–10675 (2005).

    Article  CAS  Google Scholar 

  25. Jewell, J.B., Coutinho, J.B. & Kropinski, A.M. Bioconversion of propionic, valeric, and 4-hydroxybutyric acids into the corresponding alcohols by Clostridium acetobutylicum NRRL 527. Curr. Microbiol. 13, 215–219 (1986).

    Article  CAS  Google Scholar 

  26. Nair, R.V., Bennett, G.N. & Papoutsakis, E.T. Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824. J. Bacteriol. 176, 871–885 (1994).

    Article  CAS  Google Scholar 

  27. Sivaraman, K., Seshasayee, A., Tarwater, P.M. & Cole, A.M. Codon choice in genes depends on flanking sequence information–implications for theoretical reverse translation. Nucleic Acids Res. 36, e16 (2008).

    Article  Google Scholar 

  28. Scherf, U. & Buckel, W. Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum. Appl. Environ. Microbiol. 57, 2699–2702 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Burgard, A.P., Pharkya, P. & Maranas, C.D. OptKnock: a bilevel programming framework for identifying gene knockout strategies for microbial strain optimization. Biotechnol. Bioeng. 84, 647–657 (2003).

    Article  CAS  Google Scholar 

  30. Snoep, J.L. et al. Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: implications for their activity in vivo. FEMS Microbiol. Lett. 114, 279–283 (1993).

    Article  CAS  Google Scholar 

  31. Kim, Y., Ingram, L.O. & Shanmugam, K.T. Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12. J. Bacteriol. 190, 3851–3858 (2008).

    Article  CAS  Google Scholar 

  32. Menzel, K., Zeng, A.P. & Deckwer, W.D. Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae. J. Biotechnol. 56, 135–142 (1997).

    Article  CAS  Google Scholar 

  33. Iuchi, S. & Lin, E.C. arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways. Proc. Natl. Acad. Sci. USA 85, 1888–1892 (1988).

    Article  CAS  Google Scholar 

  34. Stokell, D.J. et al. Probing the roles of key residues in the unique regulatory NADH binding site of type II citrate synthase of Escherichia coli. J. Biol. Chem. 278, 35435–35443 (2003).

    Article  CAS  Google Scholar 

  35. Pereira, D.S., Donald, L.J., Hosfield, D.J. & Duckworth, H.W. Active site mutants of Escherichia coli citrate synthase. Effects of mutations on catalytic and allosteric properties. J. Biol. Chem. 269, 412–417 (1994).

    CAS  PubMed  Google Scholar 

  36. Toth, J., Ismaiel, A.A. & Chen, J.S. The ald gene, encoding a coenzyme A-acylating aldehyde dehydrogenase, distinguishes Clostridium beijerinckii and two other solvent-producing clostridia from Clostridium acetobutylicum. Appl. Environ. Microbiol. 65, 4973–4980 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Riley, M. et al. Escherichia coli K-12: a cooperatively developed annotation snapshot–2005. Nucleic Acids Res. 34, 1–9 (2006).

    Article  CAS  Google Scholar 

  38. Orencio-Trejo, M. et al. Metabolic regulation analysis of an ethanologenic Escherichia coli strain based on RT-PCR and enzymatic activities. Biotechnol. Biofuels. 1, 8 (2008).

    Article  Google Scholar 

  39. Sprenger, G.A. & Lengeler, J.W. Analysis of sucrose catabolism in Klebsiella pneumoniae and in Scr+ derivatives of Escherichia coli K12. J. Gen. Microbiol. 134, 1635–1644 (1988).

    CAS  PubMed  Google Scholar 

  40. Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1–I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997).

    Article  CAS  Google Scholar 

  41. Donnelly, M.I., Millard, C.S., Clark, D.P., Chen, M.J. & Rathke, J.W. A novel fermentation pathway in an Escherichia coli mutant producing succinic acid, acetic acid, and ethanol. Appl. Biochem. Biotechnol. 70–72, 187–198 (1998).

    Article  Google Scholar 

  42. Kim, Y., Ingram, L.O. & Shanmugam, K.T. Construction of an Escherichia coli K-12 mutant for homoethanologenic fermentation of glucose or xylose without foreign genes. Appl. Environ. Microbiol. 73, 1766–1771 (2007).

    Article  CAS  Google Scholar 

  43. Mahadevan, R., Burgard, A., Famili, I., Van Dien, S. & Schilling, C. Applications of metabolic modeling to drive bioprocess development for the production of value-added chemicals. Biotechnol. Bioprocess Eng. 10, 408–417 (2005).

    Article  CAS  Google Scholar 

  44. Sambrook, J., Fritsh, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).

  45. Webb, E.C. Enzyme Nomenclature 1992: Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes (Academic Press, San Diego, 1992).

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Vector backbones were obtained from R. Lutz (Expressys). We thank Florida Crystals, Cargill, Bluefire Ethanol, Gruppo M&G and Verenium for providing crude sugar and biomass hydrolysate, B. Palsson, J. Keasling and G. Church for scientific advice throughout the project, and C. Schilling for critical reading of the manuscript.

Author information

Authors and Affiliations



H.Y., R.H. and W.N. cloned and expressed BDO-pathway genes, performed bottle experiments and wrote the manuscript; C.P.-B. constructed the host strain and wrote the manuscript; A.B. conceived the project, performed simulations and wrote the manuscript; J.B. cloned and expressed BDO-pathway genes; J.K. and R.S. performed analytical work; J.D.T. conceived the project and performed simulations; R.E.O. performed simulations and wrote the manuscript; J.E. constructed the host strain and performed bottle experiments; S.T. and H.B.S. performed fermentations; S.A. developed and carried out enzyme assays; T.H.Y. analyzed 13C data for flux analysis; S.Y.L. conceived the project and wrote the manuscript; M.J.B. and S.V.D. conceived and directed the project and wrote the manuscript.

Corresponding author

Correspondence to Stephen Van Dien.

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Competing interests

Support for this work was provided by Genomatica, a for-profit company pursuing commercialization of the 1,4-butanediol process discussed here. All authors except S.Y.L. were employees of Genomatica at the time the work was performed. S.Y.L. is on the scientific advisory board of Genomatica.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Methods (PDF 748 kb)

Supplementary Data 1

List of reactions included in the genome-scale E. coli model used for OptKnock analysis. (XLS 261 kb)

Supplementary Data 2

Metabolite abbreviations and names, also indicating cytosolic or extracellular location. (XLS 89 kb)

Supplementary Data 3

OptKnock results. (XLS 34 kb)

Supplementary Data 4

Set of reaction operators used by the Biopathway Predictor algorithm and corresponding reaction diagrams. (XLSX 1670 kb)

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Yim, H., Haselbeck, R., Niu, W. et al. Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat Chem Biol 7, 445–452 (2011).

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