Applied Microbiology and Biotechnology

, Volume 95, Issue 4, pp 1083–1094 | Cite as

Elucidating and reprogramming Escherichia coli metabolisms for obligate anaerobic n-butanol and isobutanol production

  • Cong T. TrinhEmail author
Bioenergy and biofuels


Elementary mode (EM) analysis based on the constraint-based metabolic network modeling was applied to elucidate and compare complex fermentative metabolisms of Escherichia coli for obligate anaerobic production of n-butanol and isobutanol. The result shows that the n-butanol fermentative metabolism was NADH-deficient, while the isobutanol fermentative metabolism was NADH redundant. E. coli could grow and produce n-butanol anaerobically as the sole fermentative product but not achieve the maximum theoretical n-butanol yield. In contrast, for the isobutanol fermentative metabolism, E. coli was required to couple with either ethanol- or succinate-producing pathway to recycle NADH. To overcome these “defective” metabolisms, EM analysis was implemented to reprogram the native fermentative metabolism of E. coli for optimized anaerobic production of n-butanol and isobutanol through multiple gene deletion (∼8–9 genes), addition (∼6–7 genes), up- and downexpression (∼6–7 genes), and cofactor engineering (e.g., NADH, NADPH). The designed strains were forced to couple both growth and anaerobic production of n-butanol and isobutanol, which is a useful characteristic to enhance biofuel production and tolerance through metabolic pathway evolution. Even though the n-butanol and isobutanol fermentative metabolisms were quite different, the designed strains could be engineered to have identical metabolic flux distribution in “core” metabolic pathways mainly supporting cell growth and maintenance. Finally, the model prediction in elucidating and reprogramming the native fermentative metabolism of E. coli for obligate anaerobic production of n-butanol and isobutanol was validated with published experimental data.


Elementary mode analysis Metabolic pathway analysis Metabolic pathway design Metabolic pathway alignment Fermentation Advanced biofuels Isobutanol n-Butanol Ethanol Rational strain design Cofactor engineering 



This research was supported in parts by the lab startup fund and the SEERC seed fund for the author at the University of Tennessee, Knoxville, TN, USA.

Supplementary material

253_2012_4197_MOESM1_ESM.pdf (409 kb)
ESM 1 (PDF 408 kb)


  1. Alper H, Stephanopoulos G (2009) Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nat Rev Micro 7:715–723CrossRefGoogle Scholar
  2. Andersch W, Bahl H, Gottschalk G (1983) Level of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum. Appl Microbiol Biotechnol 18:327–332CrossRefGoogle Scholar
  3. Atsumi S, Liao JC (2008) Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol 19:414–419CrossRefGoogle Scholar
  4. Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T, Liao JC (2008a) Metabolic engineering of Escherichia coli for 1-butanol production. Metab Eng 10:305–311CrossRefGoogle Scholar
  5. Atsumi S, Hanai T, Liao JC (2008b) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451:86–89CrossRefGoogle Scholar
  6. Bahl H, Gottwald M, Kuhn A, Rale V, Andersch W, Gottschalk G (1986) Nutritional factors affecting the ratio of solvents produced by Clostridium acetobutylicum. Appl Environ Microbiol 52:169–172Google Scholar
  7. Bastian S, Liu X, Meyerowitz JT, Snow CD, Chen MMY, Arnold FH (2011) Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli. Metab Eng 13:345–352CrossRefGoogle Scholar
  8. Berezina O, Zakharova N, Brandt A, Yarotsky S, Schwarz W, Zverlov V (2010) Reconstructing the clostridial n-butanol metabolic pathway in Lactobacillus brevis. Appl Microbiol Biotechnol 87:635–646CrossRefGoogle Scholar
  9. Berrios-Rivera SJ, Bennett GN, San K-Y (2002) Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD+-dependent formate dehydrogenase. Metab Eng 4:217–229CrossRefGoogle Scholar
  10. Blanch HW, Adams PD, Andrews-Cramer KM, Frommer WB, Simmons BA, Keasling JD (2008) Addressing the need for alternative transportation fuels: the Joint BioEnergy Institute. ACS Chem Biol 3:17–20CrossRefGoogle Scholar
  11. Boghigian BA, Shi H, Lee K, Pfeifer BA (2010) Utilizing elementary mode analysis, pathway thermodynamics, and a genetic algorithm for metabolic flux determination and optimal metabolic network design. BMC Syst Biol 4:49Google Scholar
  12. Bond-Watts BB, Bellerose RJ, Chang MCY (2011) Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat Chem Biol 7:222–227CrossRefGoogle Scholar
  13. de Graef MR, Alexeeva S, Snoep JL, Teixeira de Mattos MJ (1999) The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli. J Bacteriol 181:2351–2357Google Scholar
  14. Dellomonaco C, Clomburg JM, Miller EN, Gonzalez R (2011) Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476:355–359CrossRefGoogle Scholar
  15. Dürre P, Hollergschwandner C (2004) Initiation of endospore formation in Clostridium acetobutylicum. Anaerobe 10:69–74CrossRefGoogle Scholar
  16. Durre P, Bohringer M, Nakotte S, Schaffer S, Thormann K, Zickner B (2002) Transcriptional regulation of solventogenesis in Clostridium acetobutylicum. J Mol Microbiol Biotechnol 4:295–300Google Scholar
  17. Formanek J, Mackie R, Blaschek HP (1997) Enhanced butanol production by Clostridium beijerinckii BA101 grown in semidefined P2 medium containing 6 percent maltodextrin or glucose. Appl Environ Microbiol 63:2306–2310Google Scholar
  18. Green EM, Boynton ZL, Harris LM, Rudolph FB, Papoutsakis ET, Bennett GN (1996) Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824. Microbiology 142:2079–2086CrossRefGoogle Scholar
  19. Hädicke O, Klamt S (2011) Computing complex metabolic intervention strategies using constrained minimal cut sets. Metab Eng 13:204–213CrossRefGoogle Scholar
  20. Harris LM, Desai RP, Welker NE, Papoutsakis ET (2000) Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition? Biotechnol Bioeng 67:1–11CrossRefGoogle Scholar
  21. Hartmanis M, Gatenbeck S (1984) Intermediary metabolism in Clostridium acetobutylicum: levels of enzymes involved in the formation of acetate and butyrate. Appl Environ Microbiol 47:1277–1283Google Scholar
  22. Harvey WB (2012) Bioprocessing for biofuels. Curr Opin Biotechnol (in press)Google Scholar
  23. Herrera S (2006) Bonkers about biofuels. Nat Biotech 24:755–760CrossRefGoogle Scholar
  24. Inui M (2008) Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl Microbiol Biotechnol 77:1305–1316CrossRefGoogle Scholar
  25. Jang Y-S, Lee J, Malaviya A, Seung DY, Cho JH, Lee SY (2012) Butanol production from renewable biomass: rediscovery of metabolic pathways and metabolic engineering. Biotechnol J 7:186–198CrossRefGoogle Scholar
  26. Jevremovic D, Trinh CT, Srienc F, Boley D (2010) On algebraic properties of extreme pathways in metabolic networks. J Comp Biol 17:107–119CrossRefGoogle Scholar
  27. Jones D, Woods D (1986) Acetone-butanol fermentation revisited. Microbiol Rev 50:484–524Google Scholar
  28. Kenanov D, Kaleta C, Petzold A, Hoischen C, Diekmann S, Siddiqui RA, Schuster S (2010) Theoretical study of lipid biosynthesis in wild-type Escherichia coli and in a protoplast-type L-form using elementary flux mode analysis. FEBS 277:1023–1034CrossRefGoogle Scholar
  29. Kim Y, Ingram LO, Shanmugam KT (2008) Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12. J Bacteriol 190:3851–3858CrossRefGoogle Scholar
  30. Kromer JO, Wittmann C, Schroder H, Heinzle E (2006) Metabolic pathway analysis for rational design of L-methionine production by Escherichia coli and Corynebacterium glutamicum. Metab Eng 8:353–369CrossRefGoogle Scholar
  31. Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS (2008) Fermentative butanol production by Clostridia. Biotechnol Bioeng 101:209–228CrossRefGoogle Scholar
  32. Lee J, Jang Y-S, Choi SJ, Im JA, Song H, Cho JH, Seung DY, Papoutsakis ET, Bennett GN, Lee SY (2012) Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol–butanol–ethanol fermentation. Appl Environ Microbiol 78:1416–1423CrossRefGoogle Scholar
  33. Lehmann D, Hönicke D, Ehrenreich A, Schmidt M, Weuster-Botz D, Bahl H, Lütke-Eversloh T (2012) Modifying the product pattern of Clostridium acetobutylicum. Appl Microbiol Biotechnol 94:743–754CrossRefGoogle Scholar
  34. Long S, Jones DT, Woods DR (1983) Sporulation of Clostridium acetobutylicum P262 in a defined medium. Appl Environ Microbiol 45:1389–1393Google Scholar
  35. Martínez I, Zhu J, Lin H, Bennett GN, San K-Y (2008) Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways. Metab Eng 10:352–359CrossRefGoogle Scholar
  36. Melzer G, Esfandabadi M, Franco-Lara E, Wittmann C (2009) Flux Design: in silico design of cell factories based on correlation of pathway fluxes to desired properties. BMC Syst Biol 3:120CrossRefGoogle Scholar
  37. Mitchell WJ (1997) Physiology of carbohydrate to solvent conversion by Clostridia. In: Poole RK (ed) Advances in microbial physiology. Academic Press, New York, pp 31–130Google Scholar
  38. Mori S, Kawai S, Shi F, Mikami B, Murata K (2005) Molecular conversion of NAD kinase to NADH kinase through single amino acid residue substitution. J Biol Chem 280:24104–24112CrossRefGoogle Scholar
  39. Nielsen DR, Leonard E, Yoon SH, Tseng HC, Yuan C, Prather KL (2009) Engineering alternative butanol production platforms in heterologous bacteria. Metab Eng 11:262–273CrossRefGoogle Scholar
  40. Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, Eckert CA, Frederick WJ Jr, Hallett JP, Leak DJ, Liotta CL, Mielenz JR, Murphy R, Templer R, Tschaplinski T (2006) The path forward for biofuels and biomaterials. Science 311:484–489CrossRefGoogle Scholar
  41. Ravagnani A, Jennert KCB, Steiner E, Grünberg R, Jefferies JR, Wilkinson SR, Young DI, Tidswell EC, Brown DP, Youngman P, Morris JG, Young M (2000) Spo0A directly controls the switch from acid to solvent production in solvent-forming Clostridia. Mol Microbiol 37:1172–1185CrossRefGoogle Scholar
  42. Sauer U, Treuner A, Buchholz M, Santangelo JD, Durre P (1994) Sporulation and primary sigma factor homologous genes in Clostridium acetobutylicum. J Bacteriol 176:6572–6582Google Scholar
  43. Schubert C (2006) Can biofuels finally take center stage? Nat Biotech 24:777–784CrossRefGoogle Scholar
  44. Schuster S, Hilgetag C, Woods JH, Fell DA (1994) In: Cuthbertson R et al (eds) Elementary modes of functioning in biochemical networks. World Scientific, Singapore, pp 151–165Google Scholar
  45. Schuster S, Fell DA, Dandekar T (2000) A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic networks. Nat Biotechnol 18:326–332Google Scholar
  46. Shen CR, Lan EI, Dekishima Y, Baez A, Cho KM, Liao JC (2011) High titer anaerobic 1-butanol synthesis in Escherichia coli enabled by driving forces. Appl Environ Microbiol 77:2905–2915CrossRefGoogle Scholar
  47. Sillers R, Chow A, Tracy B, Papoutsakis ET (2008) Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance. Metab Eng 10:321–332CrossRefGoogle Scholar
  48. Steen E, Chan R, Prasad N, Myers S, Petzold C, Redding A, Ouellet M, Keasling J (2008) Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microb Cell Fact 7:36CrossRefGoogle Scholar
  49. Stelling J, Klamt S, Bettenbrock K, Schuster S, Gilles ED (2002) Metabolic network structure determines key aspects of functionality and regulation. Nature 420:190–193CrossRefGoogle Scholar
  50. Trinh CT, Srienc F (2009) Metabolic engineering of Escherichia coli for efficient conversion of glycerol to ethanol. Appl Environ Microbiol 75:6696–6705CrossRefGoogle Scholar
  51. Trinh CT, Carlson R, Wlaschin A, Srienc F (2006) Design, construction and performance of the most efficient biomass producing E. coli bacterium. Metab Eng 8:628–638CrossRefGoogle Scholar
  52. Trinh CT, Unrean P, Srienc F (2008) Minimal Escherichia coli cell for the most efficient production of ethanol from hexoses and pentoses. Appl Environ Microbiol 74:3634–3643CrossRefGoogle Scholar
  53. Trinh CT, Wlaschin A, Srienc F (2009) Elementary mode analysis: a useful metabolic pathway analysis tool for characterizing cellular metabolism. Appl Microbiol Biotechnol 81:813–826CrossRefGoogle Scholar
  54. Trinh CT, Li J, Blanch HW, Clark DS (2011) Redesigning Escherichia coli metabolism for anaerobic production of isobutanol. Appl Environ Microbiol 77:4894–4904CrossRefGoogle Scholar
  55. Unrean P, Trinh CT, Srienc F (2010) Rational design and construction of an efficient E. coli for production of diapolycopendioic acid. Metab Eng 12:112–122CrossRefGoogle Scholar
  56. von Kamp A, Schuster S (2006) Metatool 5.0: fast and flexible elementary modes analysis. Bioinformatics 22:1930–1931CrossRefGoogle Scholar
  57. Wlaschin AP, Trinh CT, Carlson R, Srienc F (2006) The fractional contributions of elementary modes to the metabolism of Escherichia coli and their estimation from reaction entropies. Metab Eng 8:338–352CrossRefGoogle Scholar
  58. Yomano L, York S, Shanmugam K, Ingram L (2009) Deletion of methylglyoxal synthase gene (mgsA) increased sugar co-metabolism in ethanol-producing Escherichia coli. Biotechnol Lett 31:1389–1398CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringUniversity of TennesseeKnoxvilleUSA

Personalised recommendations