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

• Published in: Journal of industrial microbiology & biotechnology 2012, Vol.39 (4), p.629-639
• Main Authors: Turner, Peter C., Yomano, Lorraine P., Jarboe, Laura R., York, Sean W., Baggett, Christy L., Moritz, Brélan E., Zentz, Emily B., Shanmugam, K. T., Ingram, Lonnie O.
• Format: Article
• Language: English
• Subjects: Biochemistry; Bioinformatics; Biomedical and Life Sciences; Biotechnology; Genetic Engineering; Genetics and Molecular Biology of Industrial Organisms; Inorganic Chemistry; Life Sciences; Microbiology
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-011-1052-2

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 Nco I 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 Afl II and with Bam HI 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 IS 10 , which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS 10 , 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.