Applied Microbiology and Biotechnology

, Volume 77, Issue 6, pp 1219–1224 | Cite as

Production of isopropanol by metabolically engineered Escherichia coli

  • Toru Jojima
  • Masayuki Inui
  • Hideaki YukawaEmail author
Biotechnological Products and Process Engineering


A genetically engineered strain of Escherichia coli JM109 harboring the isopropanol-producing pathway consisting of five genes encoding four enzymes, thiolase, coenzyme A (CoA) transferase, acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824, and primary–secondary alcohol dehydrogenase from C. beijerinckii NRRL B593, produced up to 227 mM of isopropanol from glucose under aerobic fed-batch culture conditions. Acetate production by the engineered strain was approximately one sixth that produced by a control E. coli strain bearing an expression vector without the clostridial genes. These results demonstrate a functional isopropanol-producing pathway in E. coli and consequently carbon flux from acetyl-CoA directed to isopropanol instead of acetate. This is the first report on isopropanol production by genetically engineered microorganism under aerobic culture conditions.


Isopropanol  Escherichia coli Clostridium acetobutylicum Clostridium beijerinckii Metabolic engineering 



We thank C. A. Omumasaba (RITE) for helpful comments on the manuscript.


  1. 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
  2. Cary JW, Petersen DJ, Papoutsakis ET, Bennett GN (1990) Cloning and expression of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli. Appl Environ Microbiol 56:1576–1583Google Scholar
  3. Chen JS, Hiu SF (1986) Acetone-butanol-isopropanol production by Clostridium beijerinckii (synonym, Clostridium butylicum). Biotechnol Lett 8:371–376CrossRefGoogle Scholar
  4. Dürre P (1998) New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation. Appl Microbiol Biotechnol 49:639–648CrossRefGoogle Scholar
  5. El-Mansi EM, Holms WH (1989) Control of carbon flux to acetate excretion during growth of Escherichia coli in batch and continuous cultures. J Gen Microbiol 135:2875–2883Google Scholar
  6. George HA, Johnson JL, Moore WEC, Holdeman LV, Chen JS (1983) Acetone, isopropanol, and butanol production by Clostridium beijerinckii (syn. Clostridium butylicum) and Clostridium aurantibutyricum. Appl Environ Microbiol 45:1160–1163Google Scholar
  7. Groot WJ, Luyben K (1986) In situ product recovery by adsorption in the butanol/isopropanol batch fermentation. Appl Micro Biotech 25:29–31Google Scholar
  8. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68CrossRefGoogle Scholar
  9. Ismaiel AA, Zhu CX, Colby GD, Chen JS (1993) Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii. J Bacteriol 175:5097–5105Google Scholar
  10. Jensen EB, Carlsen S (1990) Production of human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts. Biotechnol Bioeng 36:1–11CrossRefGoogle Scholar
  11. Krouwel PG, Groot WJ, Kossen NWF, van der Laan WFM (1983) Continuous isopropanol–butanol–ethanol fermentation by immobilized Clostridium beijerinckii cells in a packed bed fermenter. Enzyme Microbial Technol 5:46–54CrossRefGoogle Scholar
  12. Luli GW, Strohl WR (1990) Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl Environ Microbiol 56:1004–1011Google Scholar
  13. Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218CrossRefGoogle Scholar
  14. Matsumura M, Takehara S, Kataoka H (1992) Continuous butanol/isopropanol fermentation in down-flow column reactor coupled with pervaporation using supported liquid membrane. Biotechnol Bioeng 39:148–156CrossRefGoogle Scholar
  15. Mitchell W (1998) Physiology of carbohydrate to solvent conversion by clostridia. Adv Microb Physiol 39:31–130CrossRefGoogle Scholar
  16. Petersen DJ, Bennett GN (1990) Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli. Appl Environ Microbiol 56:3491–3498Google Scholar
  17. Petersen DJ, Bennett GN (1991) Cloning of the Clostridium acetobutylicum ATCC 824 acetyl coenzyme A acetyltransferase (thiolase; EC gene. Appl Environ Microbiol 57:2735–2741Google Scholar
  18. Sarmbrook J, Russell D (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, NYGoogle Scholar
  19. Yasuda K, Jojima T, Suda M, Okino S, Inui M, Yukawa H (2007) Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions. Appl Microbiol Biotechnol (in press). DOI  10.1007/s00253-007-1199-y

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Research Institute of Innovative Technology for the Earth (RITE)KizugawaJapan

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