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

Abstract

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.

Evolution of a Self-Inducible Cytolethal Distending Toxin Type V-Encoding Bacteriophage from Escherichia coli O157:H7 to Shigella sonnei

“…3). Its integration into the S. sonnei 866 lysogen was determined by designing primers attP1 and attP2 in both genes of phage DNA (Table 1) and combining these with primers attB1 and attB2 located in the fieF and cpxP genes, which were previously described as the insertion site of P2-like phages in E. coli W, E. coli K KO11FL (28), and S. sonnei 53G (accession number HE616528.1). Amplification of attP1/B1 and attB2/P2 showed amplimers of 654 and 585 bp, respectively, corresponding to the expected size and confirming that the phage was inserted into this bacterial site.…”

Polyamine Transporters and Polyamines Increase Furfural Tolerance during Xylose Fermentation with Ethanologenic Escherichia coli Strain LY180

“…We have previously identified multiple chromosomal copies of genes by examining chromosomal coverage using DNA sequence reads (35). Illumina sequence reads for EMFR35 were mapped and both genes (strain RG102).…”

Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals

“…Strain LY180 is derived from E. coli KO11, a sequenced strain that has acquired many mutations during laboratory selections for growth in mixed sugars, high sugars, lactate resistance, and other conditions (24)(25)(26). It is possible that some of the mutations in KO11 or the heterologous genes encoding ethanol production in this strain may be critical for engineering furfural tolerance and improving resistance to hemicellulose hydrolysate.…”