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)


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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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