Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes

  • Peter C. Turner
  • Lorraine P. Yomano
  • Laura R. Jarboe
  • Sean W. York
  • Christy L. Baggett
  • Brélan E. Moritz
  • Emily B. Zentz
  • K. T. Shanmugam
  • Lonnie O. Ingram
Genetics and Molecular Biology of Industrial Organisms


Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilis pdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5 kb) and pRK2 (5.4 kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.


Optical mapping Fermentation Ethanol Escherichia coli Genome sequencing 



The authors thank Savita Shanker at the DNA Sequencing core at the University of Florida for Sanger sequencing of plasmids and PCR products, and Dibyendu Kumar at the UF Bacterial Genome Finishing Program for assistance with bridging gaps between contigs. We acknowledge research support by grants from the US Department of Energy (DE-FG36-08GO88142), US Department of Agriculture, National Institute of Food and Agriculture (2011-10006-30358), and Myriant Technologies. L.O. Ingram is a consultant for Myriant Technologies and a minor stock holder (less than 4%).

Supplementary material

10295_2011_1052_MOESM1_ESM.xlsx (25 kb)
Genes present in E.coli W but deleted in KO11FL. Supplementary material 1 (XLSX 24 kb)
10295_2011_1052_MOESM2_ESM.xlsx (26 kb)
SNPs/indels present in KO11FL compared with E. coli W. Supplementary material 2 (XLSX 25 kb)
10295_2011_1052_MOESM3_ESM.pptx (78 kb)
Functional groups for all W genes and for genes deleted in KO11FL. All E. coli W genes with assigned functions, and W genes absent from KO11FL were placed in functional groups, and displayed as pie charts. Prophage genes were preferentially deleted in KO11FL, followed by genes of unknown function and transporter genes. Supplementary material 3 (PPTX 78 kb)


  1. 1.
    Archer CT, Kim JF, Jeong H, Park JH, Vickers CE, Lee SY, Nielsen LK (2011) The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli. BMC Genomics 12:9PubMedCrossRefGoogle Scholar
  2. 2.
    Barrick JE, Yu DS, Yoon SH, Jeong H, Oh TK, Schneider D, Lenski RE, Kim JF (2009) Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature 461:1243–1247PubMedCrossRefGoogle Scholar
  3. 3.
    Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1462PubMedCrossRefGoogle Scholar
  4. 4.
    Burkholder PR (1951) Determination of vitamin B12 with a mutant strain of Escherichia coli. Science 114:459–460PubMedCrossRefGoogle Scholar
  5. 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 U S A 100:825–832PubMedCrossRefGoogle Scholar
  6. 6.
    Chao L, McBroom SM (1985) Evolution of transposable elements: an IS10 insertion increases fitness in Escherichia coli. Mol Biol Evol 2:359–369PubMedGoogle Scholar
  7. 7.
    Chaudhuri RR, Loman NJ, Snyder LA, Bailey CM, Stekel DJ, Pallen MJ (2008) xBASE2: a comprehensive resource for comparative bacterial genomics. Nucleic Acids Res 36:D543–D546PubMedCrossRefGoogle Scholar
  8. 8.
    Chou HH, Berthet J, Marx CJ (2009) Fast growth increases the selective advantage of a mutation arising recurrently during evolution under metal limitation. PLoS Genet 5:e1000652PubMedCrossRefGoogle Scholar
  9. 9.
    Conrad TM, Joyce AR, Applebee MK, Barrett CL, Xie B, Gao Y, Palsson BO (2009) Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolution in lactate minimal media reveals flexible selection of adaptive mutations. Genome Biol 10:R118PubMedCrossRefGoogle Scholar
  10. 10.
    Darling AE, Mau B, Perna NT (2010) Progressive mauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One 5:e11147PubMedCrossRefGoogle Scholar
  11. 11.
    Diaz E, Ferrandez A, Prieto MA, Garcia JL (2001) Biodegradation of aromatic compounds by Escherichia coli. Microbiol Mol Biol Rev 65:523–569Google Scholar
  12. 12.
    Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2010) Geneious v5.3. Available from http://www.geneious.com
  13. 13.
    Geddes CC, Nieves IU, Ingram LO (2011) Advances in ethanol production. Curr Opin Biotechnol 22:312–319PubMedCrossRefGoogle Scholar
  14. 14.
    Gordon DM, Clermont O, Tolley H, Denamur E (2008) Assigning Escherichia coli strains to phylogenetic groups: multi-locus sequence typing versus the PCR triplex method. Environ Microbiol 10:2484–2496PubMedCrossRefGoogle Scholar
  15. 15.
    Herring CD, Raghunathan A, Honisch C, Patel T, Applebee MK, Joyce AR, Albert TJ, Blattner FR, van den Boom D, Cantor CR, Palsson BO (2006) Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale. Nat Genet 38:1406–1412PubMedCrossRefGoogle Scholar
  16. 16.
    Ingram LO, Conway T, Clark DP, Sewell GW, Preston JF (1987) Genetic engineering of ethanol production in Escherichia coli. Appl Environ Microbiol 53:2420–2425PubMedGoogle Scholar
  17. 17.
    Jarboe LR, Grabar TB, Yomano LP, Shanmugan KT, Ingram LO (2007) Development of ethanologenic bacteria. Adv Biochem Eng Biotechnol 108:237–261PubMedGoogle Scholar
  18. 18.
    Jarboe LR, Zhang X, Wang X, Moore JC, Shanmugam KT, Ingram LO (2010) Metabolic engineering for production of biorenewable fuels and chemicals: contributions of synthetic biology. J Biomed Biotechnol 2010:761042PubMedCrossRefGoogle Scholar
  19. 19.
    Keseler IM, Collado-Vides J, Santos-Zavaleta A, Peralta-Gil M, Gama-Castro S, Muniz-Rascado L, Bonavides-Martinez C, Paley S, Krummenacker M, Altman T, Kaipa P, Spaulding A, Pacheco J, Latendresse M, Fulcher C, Sarker M, Shearer AG, Mackie A, Paulsen I, Gunsalus RP, Karp PD (2011) EcoCyc: a comprehensive database of Escherichia coli biology. Nucleic Acids Res 39:D583–D590PubMedCrossRefGoogle Scholar
  20. 20.
    Kleckner N (1981) Transposable elements in prokaryotes. Annu Rev Genet 15:341–404PubMedCrossRefGoogle Scholar
  21. 21.
    Latreille P, Norton S, Goldman BS, Henkhaus J, Miller N, Barbazuk B, Bode HB, Darby C, Du Z, Forst S, Gaudriault S, Goodner B, Goodrich-Blair H, Slater S (2007) Optical mapping as a routine tool for bacterial genome sequence finishing. BMC Genomics 8:321PubMedCrossRefGoogle Scholar
  22. 22.
    Lee SY, Chang HN (1993) High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnol Lett 15:971–974CrossRefGoogle Scholar
  23. 23.
    Lenski RE, Mongold JA, Sniegowski PD, Travisano M, Vasi F, Gerrish PJ, Schmidt TM (1998) Evolution of competitive fitness in experimental populations of E. coli: what makes one genotype a better competitor than another? Antonie Van Leeuwenhoek 73:35–47PubMedCrossRefGoogle Scholar
  24. 24.
    Miller EN, Jarboe LR, Yomano LP, York SW, Shanmugam KT, Ingram LO (2009) Silencing of NADPH-dependent oxidoreductase genes (yqhD and dkgA) in furfural-resistant ethanologenic Escherichia coli. Appl Environ Microbiol 75:4315–4323PubMedCrossRefGoogle Scholar
  25. 25.
    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
  26. 26.
    Oshima K, Toh H, Ogura Y, Sasamoto H, Morita H, Park SH, Ooka T, Iyoda S, Taylor TD, Hayashi T, Itoh K, Hattori M (2008) Complete genome sequence and comparative analysis of the wild-type commensal Escherichia coli strain SE11 isolated from a healthy adult. DNA Res 15:375–386PubMedCrossRefGoogle Scholar
  27. 27.
    Ross DG, Swan J, Kleckner N (1979) Nearly precise excision: a new type of DNA alteration associated with the translocatable element Tn10. Cell 16:733–738PubMedCrossRefGoogle Scholar
  28. 28.
    Schumacher G, Sizmann D, Haug H, Buckel P, Bock A (1986) Penicillin acylase from E. coli: unique gene-protein relation. Nucleic Acids Res 14:5713–5727PubMedCrossRefGoogle Scholar
  29. 29.
    Schwan WR, Briska A, Stahl B, Wagner TK, Zentz E, Henkhaus J, Lovrich SD, Agger WA, Callister SM, DuChateau B, Dykes CW (2010) Use of optical mapping to sort uropathogenic Escherichia coli strains into distinct subgroups. Microbiology 156:2124–2135PubMedCrossRefGoogle Scholar
  30. 30.
    Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M (2006) ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res 34:D32–D36PubMedCrossRefGoogle Scholar
  31. 31.
    Sobotkova L, Grafkova J, Stepanek V, Vacik T, Maresova H, Kyslik P (1999) Indigenous plasmids in a production line of strains for penicillin G acylase derived from Escherichia coli W. Folia Microbiol (Praha) 44:263–266CrossRefGoogle Scholar
  32. 32.
    Stoebel DM, Dorman CJ (2010) The effect of mobile element IS10 on experimental regulatory evolution in Escherichia coli. Mol Biol Evol 27:2105–2112PubMedCrossRefGoogle Scholar
  33. 33.
    Stoebel DM, Hokamp K, Last MS, Dorman CJ (2009) Compensatory evolution of gene regulation in response to stress by Escherichia coli lacking RpoS. PLoS Genet 5:e1000671PubMedCrossRefGoogle Scholar
  34. 34.
    Touchon M, Hoede C, Tenaillon O, Barbe V, Baeriswyl S, Bidet P, Bingen E, Bonacorsi S, Bouchier C, Bouvet O, Calteau A, Chiapello H, Clermont O, Cruveiller S, Danchin A, Diard M, Dossat C, Karoui ME, Frapy E, Garry L, Ghigo JM, Gilles AM, Johnson J, Le Bouguenec C, Lescat M, Mangenot S, Martinez-Jehanne V, Matic I, Nassif X, Oztas S, Petit MA, Pichon C, Rouy Z, Ruf CS, Schneider D, Tourret J, Vacherie B, Vallenet D, Medigue C, Rocha EP, Denamur E (2009) Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths. PLoS Genet 5:e1000344PubMedCrossRefGoogle Scholar
  35. 35.
    Turner PC, Miller EN, Jarboe LR, Baggett CL, Shanmugam KT, Ingram LO (2010) YqhC regulates transcription of the adjacent Escherichia coli genes yqhD and dkgA that are involved in furfural tolerance. J Ind Microbiol Biotechnol 38:431–439PubMedCrossRefGoogle Scholar
  36. 36.
    Wang X, Miller EN, Yomano LP, Zhang X, Shanmugam KT, Ingram LO (2011) Overexpression of NADH-dependent oxidoreductase fucO increases furfural tolerance in Escherichia coli strains engineered for the production of ethanol and lactate. Appl Environ Microbiol 77:5132–5140PubMedCrossRefGoogle Scholar
  37. 37.
    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 Biotechnol 20:132–138PubMedCrossRefGoogle Scholar
  38. 38.
    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 31:1389–1398PubMedCrossRefGoogle Scholar
  39. 39.
    Yomano LP, York SW, Zhou S, Shanmugam KT, Ingram LO (2008) Re-engineering Escherichia coli for ethanol production. Biotechnol Lett 30:2097–2103PubMedCrossRefGoogle Scholar
  40. 40.
    Zhang X, Jantama K, Shanmugam KT, Ingram LO (2009) Reengineering Escherichia coli for succinate production in mineral salts medium. Appl Environ Microbiol 75:7807–7813PubMedCrossRefGoogle Scholar
  41. 41.
    Zhang X, Wang X, Shanmugam KT, Ingram LO (2011) l-Malate production by metabolically engineered Escherichia coli. Appl Environ Microbiol 77:427–434PubMedCrossRefGoogle Scholar
  42. 42.
    Zhang X, Jantama K, Moore JC, Shanmugam KT, Ingram LO (2007) Production of l-alanine by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 77:355–366PubMedCrossRefGoogle Scholar
  43. 43.
    Zhang X, Jantama K, Moore JC, Jarboe LR, Shanmugam KT, Ingram LO (2009) Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli. Proc Natl Acad Sci U S A 106:20180–20185PubMedCrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2011

Authors and Affiliations

  • Peter C. Turner
    • 1
  • Lorraine P. Yomano
    • 1
  • Laura R. Jarboe
    • 1
    • 3
  • Sean W. York
    • 1
  • Christy L. Baggett
    • 1
  • Brélan E. Moritz
    • 1
  • Emily B. Zentz
    • 2
  • K. T. Shanmugam
    • 1
  • Lonnie O. Ingram
    • 1
  1. 1.Department of Microbiology and Cell BiologyUniversity of FloridaGainesvilleUSA
  2. 2.OpGen Inc.GaithersburgUSA
  3. 3.Chemical and Biological Engineering, Biorenewables Research LaboratoryIowa State UniversityAmesUSA

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