Advertisement

Biotechnology Letters

, Volume 30, Issue 12, pp 2097–2103 | Cite as

Re-engineering Escherichia coli for ethanol production

  • L. P. Yomano
  • S. W. York
  • S. Zhou
  • K. T. Shanmugam
  • L. O. IngramEmail author
Original Research Paper

Abstract

A lactate producing derivative of Escherichia coli KO11, strain SZ110, was re-engineered for ethanol production by deleting genes encoding all fermentative routes for NADH and randomly inserting a promoterless mini-Tn5 cassette (transpososome) containing the complete Zymomonas mobilis ethanol pathway (pdc, adhA, and adhB) into the chromosome. By selecting for fermentative growth in mineral salts medium containing xylose, a highly productive strain was isolated in which the ethanol cassette had been integrated behind the rrlE promoter, designated strain LY160 (KO11, Δfrd::celY Ec ΔadhE ΔldhA, ΔackA lacA::casAB Ko rrlE::(pdc Zm -adhA Zm -adhB Zm -FRT-rrlE) pflB + ). This strain fermented 9% (w/v) xylose to 4% (w/v) ethanol in 48 h in mineral salts medium, nearly equal to the performance of KO11 with Luria broth.

Keywords

Betaine E. coli Ethanol Lactate Osmotic stress 

Notes

Acknowledgements

This research was supported by grants from the U.S. Department of Agriculture (01-35504-10669 and 00-52104-9704), the U.S. Department of Energy (FG02-96ER20222 and FG36-04GO14019), and by the Verenium Corporation.

References

  1. Alterthum F, Ingram LO (1989) Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli. Appl Environ Microbiol 55:1943–1948PubMedGoogle Scholar
  2. Arntzen CE, Dale BE (1999) Biobased industrial products priorities for research and commercialization. National Academy Press, Washington, DCGoogle Scholar
  3. Asghari A, Bothast RJ, Doran JB, Ingram LO (1996) Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineered Escherichia coli. J Ind Microbiol 16:42–47CrossRefGoogle Scholar
  4. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) (1987) Current protocols in molecular biology. Wiley, Inc, New YorkGoogle Scholar
  5. Causey TB, Zhou S, Shanmugam KT, Ingram LO (2003) Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: homoacetate production. Proc Natl Acad Sci USA 100:825–832PubMedCrossRefGoogle Scholar
  6. Conway T, Sewell GW, Osman YA, Ingram LO (1987) Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis. J Bacteriol 169:2591–2597PubMedGoogle Scholar
  7. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645PubMedCrossRefGoogle Scholar
  8. de Lorenzo V, Herrero M, Jakubzik U, Timmis KN (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172:6568–6572PubMedGoogle Scholar
  9. Grabar TB, Zou S, Shanmugam KT, Yomano LP, Ingram LO (2006) Methylglyoxal bypass identified as source of chiral contamination in l(+) and d(−)-lactate fermentations by recombinant Escherichia coli. Biotechnol Lett 28:1527–1535PubMedCrossRefGoogle Scholar
  10. Hahn-Hagerdal B, Galbe M, Gorwa-Grauslund MF, Liden G, Zacchi G (2006) Bio-ethanol—the fuel of tomorrow from the residues of today. Trends Biotechnol 24:549–556PubMedCrossRefGoogle Scholar
  11. Ingram LO, Gomez PF, Lai X, Moniruzzaman M, Wood BE, Yomano LP, York SW (1998) Metabolic engineering of bacteria for ethanol production. Biotechnol Bioeng 58:204–214PubMedCrossRefGoogle Scholar
  12. Keshav KF, Yomano LP, An H, Ingram LO (1990) Cloning of the Zymomonas mobilis structural gene encoding alcohol dehydrogenase I (adhA): sequencing comparison and expression in Escherichia coli. J Bacteriol 172:2491–2497PubMedGoogle Scholar
  13. Mackenzie KF, Eddy CK, Ingram LO (1989) Modulation of alcohol dehydrogenase isoenzyme levels in Zymomonas mobilis by iron and zinc. J Bacteriol 171:1063–1067PubMedGoogle Scholar
  14. Martinez A, York SW, Yomano LP, Pineda VL, Davis FC, Shelton JC, Ingram LO (1999) Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli. Biotechnol Prog 15:891–897PubMedCrossRefGoogle Scholar
  15. Martinez-Morales F, Borges AC, Martinez A, Shanmugam KT, Ingram LO (1999) Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons. J Bacteriol 181:7143–7148PubMedGoogle Scholar
  16. Miller JH (1992) A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  17. Ohta K, Beall DS, Mejia JP, Shanmugam KT, Ingram LO (1991) Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II. Appl Environ Microbiol 57:893–900PubMedGoogle Scholar
  18. Posfai G, Koob MD, Kirkpatrick HA, Blattner FR (1997) Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome. J Bacteriol 179:4426–4428PubMedGoogle Scholar
  19. Purvis JE, Yomano LP, Ingram LO (2005) Enhanced trehalose production improves growth of Escherichia coli under osmotic stress (salts and sugars). Appl Environ Microbiol 71:3761–3769PubMedCrossRefGoogle Scholar
  20. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  21. Underwood SA, Buszko ML, Shanmugam KT, Ingram LO (2004) Lack of protective osmolytes limits final cell density and volumetric productivity of ethanologenic Escherichia coli KO11 during xylose fermentation. Appl Environ Microbiol 70:2734–2740PubMedCrossRefGoogle Scholar
  22. Wanner BL, Kodaira R, Neidhardt FC (1977) Physiological regulation of a decontrolled lac operon. J Bacteriol 130:212–222PubMedGoogle Scholar
  23. York SW, Ingram LO (1996) Ethanol production by recombinant Escherichia coli KO11 using crude yeast autolysate as a nutrient supplement. Biotechnol Lett 18:683–688CrossRefGoogle Scholar
  24. Zhang J, Greasham R (1999) Chemically defined media for commercial fermentations. Appl Microbiol Biotechnol 51:407–421CrossRefGoogle Scholar
  25. Zhou S, Yomano LP, Shanmugam KT, Ingram LO (2005) Fermentation of 10% sugar to d(−)-lactate by engineered Escherichia coli B. Biotechnol Lett 27:1891–1896PubMedCrossRefGoogle Scholar
  26. Zhou S, Grabar TB, Shanmugam KT, Ingram LO (2006a) Betaine tripled the volumetric productivity of d(−)-lactate by Escherichia coli SZ132 in mineral salts medium. Biotechnol Lett 28:671–676PubMedCrossRefGoogle Scholar
  27. Zhou S, Shanmugam KT, Yomano LP, Grabar TB, Ingram LO (2006b) Fermentation of 12% glucose (w/v) to 1.2 M lactate by Escherichia coli strain SZ194 using mineral salts medium. Biotechnol Lett 28:663–670PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • L. P. Yomano
    • 1
  • S. W. York
    • 1
  • S. Zhou
    • 1
  • K. T. Shanmugam
    • 1
  • L. O. Ingram
    • 1
    Email author
  1. 1.Department Microbiology and Cell ScienceUniversity of FloridaGainesvilleUSA

Personalised recommendations