Applied Microbiology and Biotechnology

, Volume 86, Issue 2, pp 419–434 | Cite as

Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology



The microbial production of biofuels is a promising avenue for the development of viable processes for the generation of fuels from sustainable resources. In order to become cost and energy effective, these processes must utilize organisms that can be optimized to efficiently produce candidate fuels from a variety of feedstocks. Escherichia coli has become a promising host organism for the microbial production of biofuels in part due to the ease at which this organism can be manipulated. Advancements in metabolic engineering and synthetic biology have led to the ability to efficiently engineer E. coli as a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. This review focuses on recent efforts devoted to engineering E. coli for the production of biofuels, with emphasis on the key aspects of both the utilization of a variety of substrates as well as the synthesis of several promising biofuels. Strategies for the efficient utilization of carbohydrates, carbohydrate mixtures, and noncarbohydrate carbon sources will be discussed along with engineering efforts for the exploitation of both fermentative and nonfermentative pathways for the production of candidate biofuels such as alcohols and higher carbon biofuels derived from fatty acid and isoprenoid pathways. Continued advancements in metabolic engineering and synthetic biology will help improve not only the titers, yields, and productivities of biofuels discussed herein, but also increase the potential range of compounds that can be produced.


Synthetic biology Metabolic engineering Biofuels production Escherichia coli Biofuels 


  1. Aristidou A, Penttila M (2000) Metabolic engineering applications to renewable resource utilization. Curr Opin Biotechnol 11:187–198CrossRefGoogle Scholar
  2. 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
  3. Atsumi S, Hanai T, Liao JC (2008b) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451:86–89CrossRefGoogle Scholar
  4. Atsumi S, Liao JC (2008a) Directed evolution of Methanococcus jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli. Appl Environ Microbiol 74:7802–7808CrossRefGoogle Scholar
  5. Atsumi S, Liao JC (2008b) Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol 19:414–419CrossRefGoogle Scholar
  6. Babitzke P, Romeo T (2007) CsrB sRNA family: sequestration of RNA-binding regulatory proteins. Curr Opin Microbiol 10:156–163CrossRefGoogle Scholar
  7. Bailey JE (1991) Toward a Science of Metabolic Engineering. Science 252:1668–1675CrossRefGoogle Scholar
  8. Bermejo LL, Welker NE, Papoutsakis ET (1998) Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxification. Appl Environ Microbiol 64:1079–1085Google Scholar
  9. Cann AF, Liao JC (2008) Production of 2-methyl-1-butanol in engineered Escherichia coli. Appl Microbiol Biotechnol 81:89–98CrossRefGoogle Scholar
  10. Carothers JM, Goler JA, Keasling JD (2009) Chemical synthesis using synthetic biology. Curr Opin Biotechnol 20:498–503CrossRefGoogle Scholar
  11. Chen JS, Hiu SF (1986) Acetone butanol isopropanol production by Clostridium beijerinckii. Biotechnol Lett 8:371–376CrossRefGoogle Scholar
  12. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306CrossRefGoogle Scholar
  13. Christianson DW (2008) Unearthing the roots of the terpenome. Curr Opin Chem Biol 12:141–150CrossRefGoogle Scholar
  14. Connor MR, Liao JC (2008) Engineering of an Escherichia coli strain for the production of 3-methyl-1-butanol. Appl Environ Microbiol 74:5769–5775CrossRefGoogle Scholar
  15. Connor MR, Liao JC (2009) Microbial production of advanced transportation fuels in non-natural hosts. Curr Opin Biotechnol 20:307–315CrossRefGoogle Scholar
  16. Daniel R, Gottschalk G (1992) Growth temperature-dependent acitivity of glycerol dehydratase in Escherichia coli expressing the Citrobacter freundii dha Regulon. FEMS Microbiol Lett 100:281–285Google Scholar
  17. Decker K, Plumbridge J, Boos W (1998) Negative transcriptional regulation of a positive regulator: the expression of malT, encoding the transcriptional activator of the maltose regulon of Escherichia coli, is negatively controlled by Mlc. Mol Microbiol 27:381–390CrossRefGoogle Scholar
  18. Dewick PM (2002) The biosynthesis of C-5-C-25 terpenoid compounds. Nat Prod Rep 19:181–222CrossRefGoogle Scholar
  19. Dharmadi Y, Murarka A, Gonzalez R (2006) Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering. Biotechnol Bioeng 94:821–829CrossRefGoogle Scholar
  20. Doan TTP, Carlsson AS, Hamberg M, Bulow L, Stymne S, Olsson P (2009) Functional expression of five Arabidopsis fatty acyl-CoA reductase genes in Escherichia coli. J Plant Physiol 166:787–796CrossRefGoogle Scholar
  21. Durnin G, Clomburg J, Yeates Z, Alvarez PJJ, Zygourakis K, Campbell P, Gonzalez R (2009) Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli. Biotechnol Bioeng 103:148–161CrossRefGoogle Scholar
  22. Durrett TP, Benning C, Ohlrogge J (2008) Plant triacylglycerols as feedstocks for the production of biofuels. Plant J 54:593–607CrossRefGoogle Scholar
  23. Ezeji TC, Qureshi N, Blaschek HP (2005) Continuous butanol fermentation and feed starch retrogradation: butanol fermentation sustainability using Clostridium beijerinckii BA101. J Biotechnol 115:179–187CrossRefGoogle Scholar
  24. Fischer CR, Klein-Marcuschamer D, Stephanopoulos G (2008) Selection and optimization of microbial hosts for biofuels production. Metab Eng 10:295–304CrossRefGoogle Scholar
  25. Flores N, Xiao J, Berry A, Bolivar F, Valle F (1996) Pathway engineering for the production of aromatic compounds in Escherichia coli. Nat Biotechnol 14:620–623CrossRefGoogle Scholar
  26. Fortman JL, Chhabra S, Mukhopadhyay A, Chou H, Lee TS, Steen E, Keasling JD (2008) Biofuel alternatives to ethanol: pumping the microbial well. Trends Biotechnol 26:375–381CrossRefGoogle Scholar
  27. Friedman L, Rude M (2008) Process for producing low molecular weight hydrocarbons from renewable resources. WO/2008/113041Google Scholar
  28. Goerke B, Stulke J (2008) Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol 6:613–624CrossRefGoogle Scholar
  29. Gonzalez R, Campbell P, Wong M (2009) Production of ethanol from thin stillage by metabolically engineered Escherichia coli. Biotechnol Lett 32. doi:10.1007/s10529-009-0159-2 Google Scholar
  30. Gonzalez R, Murarka A, Dharmadi Y, Yazdani SS (2008) A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli. Metab Eng 10:234–245CrossRefGoogle Scholar
  31. Gonzalez R, Tao H, Purvis JE, York SW, Shanmugam KT, Ingram LO (2003) Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: Comparison of KO11 (Parent) to LY01 (resistant mutant). Biotechnol Prog 19:612–623CrossRefGoogle Scholar
  32. Hanai T, Atsumi S, Liao JC (2007) Engineered synthetic pathway for isopropanol production in Escherichia coli. Appl Environ Microbiol 73:7814–7818CrossRefGoogle Scholar
  33. Hardiman T, Lemuth K, Keller MA, Reuss M, Siemann-Herzberg M (2007) Topology of the global regulatory network of carbon limitation in Escherichia coli. J Biotechnol 132:359–374CrossRefGoogle Scholar
  34. Hernandez-Montalvo V, Martinez A, Hernandez-Chavez G, Bolivar F, Valle F, Gosset G (2003) Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products. Biotechnol Bioeng 83:687–694CrossRefGoogle Scholar
  35. Hernandez-Montalvo V, Valle F, Bolivar F, Gosset G (2001) Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system. Appl Microbiol Biotechnol 57:186–191CrossRefGoogle Scholar
  36. Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315:804–807CrossRefGoogle Scholar
  37. Hirsch RL, Bezdek R, Wendling R (2006) Peaking of world oil production and its mitigation. AIChE J 52:2–8CrossRefGoogle Scholar
  38. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54:621–639CrossRefGoogle Scholar
  39. Ideker T, Galitski T, Hood L (2001) A new approach to decoding life: systems biology. Annu Rev Genomics Hum Genet 2:343–372CrossRefGoogle Scholar
  40. Ingram LO, Conway T, Clark DP, Sewell GW, Preston JF (1987) Genetic engineering of ethanol production in Escherichia coli. Appl Environ Microbiol 53:2420–2425Google Scholar
  41. Inui M, Suda M, Kimura S, Yasuda K, Suzuki H, Toda H, Yamamoto S, Okino S, Suzuki N, Yukawa H (2008) Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl Microbiol Biotechnol 77:1305–1316CrossRefGoogle Scholar
  42. Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited. Microbiol Rev 50:484–524Google Scholar
  43. Kalscheuer R, Stolting T, Steinbuchel A (2006) Microdiesel: Escherichia coli engineered for fuel production. Microbiology 152:2529–2536CrossRefGoogle Scholar
  44. Keasling JD, Hu Z, Somerville C, Church G, Berry D, Friedman L, Schirmer A, Brubaker S, Del Cardayre SB (2007) Production of fatty acids and derivatives thereof. WO/2007/136762Google Scholar
  45. Kerr RA (2007) Global warming is changing the world. Science 316:188–190CrossRefGoogle Scholar
  46. Khankal R, Chin JW, Ghosh D, Cirino PC (2009) Transcriptional effects of CRP* expression in Escherichia coli. J Biol Eng 3:13CrossRefGoogle Scholar
  47. Kim Y, Ingram LO, Shanmugam KT (2007) Construction of an Escherichia coli K-12 mutant for homoethanologenic fermentation of glucose or xylose without foreign genes. Appl Environ Microbiol 73:1766–1771CrossRefGoogle Scholar
  48. Kimata K, Inada T, Tagami H, Aiba H (1998) A global repressor (MIc) is involved in glucose induction of the ptsG gene encoding major glucose transporter in Escherichia coli. Mol Microbiol 29:1509–1519CrossRefGoogle Scholar
  49. Knoshaug EP, Zhang M (2009) Butanol tolerance in a selection of microorganisms. Appl Biochem Biotechnol 153:13–20CrossRefGoogle Scholar
  50. Lee SK, Chou H, Ham TS, Lee TS, Keasling JD (2008) Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 19:556–563CrossRefGoogle Scholar
  51. Lu XF, Vora H, Khosla C (2008) Overproduction of free fatty acids in E. coli: implications for biodiesel production. Metab Eng 10:333–339CrossRefGoogle Scholar
  52. Ma FR, Hanna MA (1999) Biodiesel production: a review. Bioresour Technol 70:1–15CrossRefGoogle Scholar
  53. Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21:796–802CrossRefGoogle Scholar
  54. Mukhopadhyay A, Redding AM, Rutherford BJ, Keasling JD (2008) Importance of systems biology in engineering microbes for biofuel production. Curr Opin Biotechnol 19:228–234CrossRefGoogle Scholar
  55. Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R (2008) Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl Environ Microbiol 74:1124–1135CrossRefGoogle Scholar
  56. Nichols NN, Dien BS, Bothast RJ (2001) Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol. Appl Microbiol Biotechnol 56:120–125CrossRefGoogle Scholar
  57. Nielsen DR, Leonard E, Yoon SH, Tseng HC, Yuan C, Jones Prather KL (2009) Engineering alternative butanol production platforms in heterologous bacteria. Metab Eng 11:262–273CrossRefGoogle Scholar
  58. Ohta K, Beall DS, Mejia JP, Shanmugam KT, Ingram LO (1991) Genetic improvement of Escherichia coli for ehtnao, production: chromosomal integration of Zymomonas mobilis genes enconding pyruvate decarboxylase and alcohol dehydrogenase II. Appl Environ Microbiol 57:893–900Google Scholar
  59. Oren A (2005) A hundred years of Dunaliella research: 1905–2005. Saline Syst 1:2CrossRefGoogle Scholar
  60. Pleiss J (2006) The promise of synthetic biology. Appl Microbiol Biotechnol 73:735–739CrossRefGoogle Scholar
  61. Rude MA, Schirmer A (2009) New microbial fuels: a biotech perspective. Curr Opin Microbiol 12:274–281CrossRefGoogle Scholar
  62. Rodriguez-Moya M, Gonzalez R (2009) Systems biology approaches for the microbial production of biofuels. Biofuels 1. doi:10.4155/BFS.10.5
  63. Sawers RG, Clark DP (2004) Fermentative pyruvate and acetyl-CoA metabolism. In: Neidhardt FC, Curtiss R III, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella: cellular and molecular biology, Webth edn. ASM Press, WashingtonGoogle Scholar
  64. Schubert C (2006) Can biofuels finally take center stage? Nat Biotechnol 24:777–784CrossRefGoogle Scholar
  65. Service RF (2009) Biofuels: ExxonMobil fuels venter’s efforts to run vehicles on algae-based oil. Science 325:379–379CrossRefGoogle Scholar
  66. Shen CR, Liao JC (2008) Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol production via the keto-acid pathways. Metab Eng 10:312–320CrossRefGoogle Scholar
  67. Skraly FA, Lytle BL, Cameron DC (1998) Construction and characterization of a 1,3-propanediol operon. Appl Environ Microbiol 64:98–105Google Scholar
  68. Stephanopoulos G (2002) Metabolic engineering: perspective of a chemical engineer. AIChE J 48:920–926CrossRefGoogle Scholar
  69. Stephanopoulos G (2007) Challenges in engineering microbes for biofuels production. Science 315:801–804CrossRefGoogle Scholar
  70. Stulke J, Hillen W (1999) Carbon catabolite repression in bacteria. Curr Opin Microbiol 2:195–201CrossRefGoogle Scholar
  71. Tao H, Gonzalez R, Martinez A, Rodriguez M, Ingram LO, Preston JF, Shanmugam KT (2001) Engineering a homo-ethanol pathway in Escherichia coli: Increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation. J Bacteriol 183:2979–2988CrossRefGoogle Scholar
  72. Totemeyer S, Booth NA, Nichols WW, Dunbar B, Booth IR (1998) From famine to feast: the role of methylglyoxal production in Escherichia coli. Mol Microbiol 27:553–562CrossRefGoogle Scholar
  73. Trinh CT, Srienc F (2009) Metabolic Engineering of Escherichia coli for Efficient Conversion of Glycerol to Ethanol. Appl Environ Microbiol 75:6696–6705CrossRefGoogle Scholar
  74. 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
  75. Withers ST, Gottlieb SS, Lieu B, Newman JD, Keasling JD (2007) Identification of isopentenol biosynthetic genes from Bacillus subtilis by a screening method based on isoprenoid precursor toxicity. Appl Environ Microbiol 73:6277–6283CrossRefGoogle Scholar
  76. Yazdani SS, Gonzalez R (2007) Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opin Biotechnol 18:213–219CrossRefGoogle Scholar
  77. Yazdani SS, Gonzalez R (2008) Engineering Escherichia coli for the efficient conversion of glycerol to ethanol and co-products. Metab Eng 10:340–351CrossRefGoogle Scholar
  78. Yomano LP, York SW, Ingram LO (1998) Isolation and characterization of ethanol-tolerant mutants of Escherichia coli KO11 for fuel ethanol production. J Ind Microbiol Biotech 20:132–138CrossRefGoogle Scholar
  79. Yomano LP, York SW, Shanmugam KT, Ingram LO (2009) Deletion of methylglyoxal synthase gene (mgsA) increased sugar co-metabolism in ethanol-producing Escherichia coli. Biotechnol Lett 31:1389–1398CrossRefGoogle Scholar
  80. Yomano LP, York SW, Zhou S, Shanmugam KT, Ingram LO (2008) Re-engineering Escherichia coli for ethanol production. Biotechnol Lett 30:2097–2103CrossRefGoogle Scholar
  81. Zhang KC, Sawaya MR, Eisenberg DS, Liao JC (2008) Expanding metabolism for biosynthesis of nonnatural alcohols. Proc Natl Acad Sci USA 105:20653–20658CrossRefGoogle Scholar
  82. Zhu MM, Skraly FA, Cameron DC (2001) Accumulation of methylglyoxal in anaerobically grown Escherichia coli and its detoxification by expression of the Pseudomonas putida glyoxalase I gene. Metab Eng 3:218–225CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringRice UniversityHoustonUSA
  2. 2.Department of BioengineeringRice UniversityHoustonUSA

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